Toronto/Lab Notebook

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<font size=4>< [[October]] | [[September]] | [[August]] ></font>
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<font size=4>< [[Toronto/Lab Notebook/August|August]] | [[Toronto/Lab Notebook/September|September]] | [[Toronto/Lab Notebook|Current]] ></font>
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-
== Oct. 1, 2007 (Monday) ==
+
[[Toronto/PartsCatalogue|Parts Catalogue]]
-
'''Time: 3:45 PM - 8:30 PM'''
+
== October 24 ==
 +
* ligate JI + N4 (AMP) into DH5a; this will be module D
 +
* ligate JI + RE (AMP) into DH5a; this will be module A
 +
* enzyme contamination test: cross-contamination detected in Xba and Spe tubes.
-
Yusuf, Irina
+
== October 23 ==
 +
* length check JI (J23100 + I0462): pass
 +
* quantitate JI, N4, Q04400B:
 +
** JI: 4.5 ng/uL
 +
** N4: 4.5 ng/uL
 +
** Q04400B: no bands seen
 +
* overnight prep of T9002(CDE), Q04400E
 +
* length check of E1A(AB): pass, but ghost bands present. Check for enzyme contamination.
-
Summary:
+
== October 20 ==
 +
(Anam)
 +
* miniprepped T9002(AB), E1A(AB)
 +
* quantitated Q04400B: no bands seen
 +
* length checks of T9002, N4, N5: all pass
-
* made 5 KAN plates
+
== October 19, 2007 ==
-
* made 2 AMP+KAN plates
+
-
* made 300 mL LB
+
-
Tranformation of following parts:
+
Yusuf, Maria, Faareha, Talal
-
* I13033 -> KAN
+
-
* E0433 -> KAN
+
-
* S03140 -> AMP
+
-
* J04500 -> KAN
+
-
* J31003 -> AMP
+
-
* (J23100C + S01640B)+ B0015 -> KAN
+
-
Used 4uL of DNA for all transformations
+
Start: 3pm - 12am
-
* tried to do DNA extraction for J06501 but somebody already extracted it (wasn't in the well)
+
'''SUMMARY:'''
-
* couldn't find it in the igem dna box as well !!!
+
* MP O/N for E1A and T9002
 +
* Transformation of J23100C and I0462 since no colonies grew
 +
* Quantitation for the following constructs (with results):
 +
** I13033 A                          - no band seen
 +
** I13504 with X/P                    - no band seen
 +
** N1A from Oct 4/07 with S/P        - 13.5ng/μL
 +
** N1A from Oct 12/07                - 13.5ng/μL
 +
** R0082                              - 5.5ng/μL
-
TO DO:
+
* Digest
 +
** Q04400 B - primary digest with X/P wasn't successful. Only plasmid was seen. Second digest with E/S
 +
*** If digest of Q04400 has been ok ed by charles proceed to Quantitation and ligation and transformation
 +
** Enzyme check for SpeI - all enzymes working
-
* MP o/n for the above parts
+
'''TO DO:'''
 +
** MP E1A and T9002
 +
** MP O/N for (J23100C + I0462)
 +
** Make AMP plates
 +
** After Charles approves it, continue to quantitation and ligation for Q04400B
 +
** Re quantitate the following I13033 and I13504 bcause no bands were seen when i did the quantitation
 +
* make competent cells, both DH5a and DH5a-z1 (Make about 12 each b/c we don't have that many tubes)
 +
* Charles will transform the pPCB and pCph8 plasmids
 +
== October 18th, 2007 ==
 +
Natalie
 +
*Rechecked Q04400B - everything is OK (verified by Seema and Esther).
-
== Sept. 30, 2007 (Sunday) ==
+
Esther, Neha, Rafsan
-
'''Time: 1:10 PM - 4:23 PM '''
+
1. MPs:
 +
*N4 (A,B) x2
 +
*N5 (A,B) x2
 +
*Stock cells of all of the above in the -80°C freezer
 +
*Some MP leftover - supernatent drained, put into -20°C freezer (if MP from today is poor, use these to redo the MP).
-
Esther
+
2. Transformation of E1b, N1 (new)
 +
*In the incubator.
 +
* Something is odd about these stock cells - they were taken from the -80°C freezer, iGEM box, labeled with "alpha" (presumably they are DH5a). Discuss at tomorrow's meeting.
-
Summary:
+
3. PD of N4 A,B and N5 A,B - used X/P
-
Done Today:  
+
4. Quantitation:
 +
*I13033
 +
*N1A
 +
*R0082 #1
 +
*I13504A
-
1. DNA extraction from plates for:
+
Note: The gel used was yesterday's.
-
*I13033, E0433, S03140, J04500, J31003 (Note: There may be a problem with this one, check notebook)
+
-
2. MP of S01640B
+
Results:
-
3. Stock cells of S01640B x 4
+
Inconclusive. I have some doubts about the integrity of the wells in the gel - some of them were stuck together, some allowed loading dye to float out, etc. As a result, I am going to ask you redo the check for all of the above using a new gel.
-
To Do:  
+
To do:  
-
1. Transform items from #1 as well as (J23100+S01640)+B0015 (in freezer).  
+
1. MP O/N of E1b, N1
 +
2. PD check: PD of N4 A,B and N5 A,B
 +
3. Quantitate:
 +
*I13033
 +
*N1A
 +
*R0082 #1
 +
*I13504A
 +
4. PD and GE of Q04400B - use the EXACT tube Natalie used yesterday - check the date (this is noted in the lab notebook).
-
2. Make LB and plates
+
== October 17th, 2007 ==
-
3. Recheck (on the computer!) the lengths for PD from Sept. 29th.
+
Yusuf (5:00pm - 10:00pm)
-
*If okay, proceed
+
-
*If not, troubleshoot
+
-
== Sept 29, 2007 ==
+
'''SUMMARY:'''
 +
*''' As optimistic as Natalie was, there is no way the parts Q04400B and I13033 match up with the bands seen in the gel. So no match once again.'''
 +
* Ligation of J23100C with I0462
 +
** the part has been named as N1 in the iGem Parts Excel sheet.  Discard all other N1 and only keep the new N1
 +
* Mini prep O/N for N4 (A,B) and N5 (A,B)
-
'''Time: 1:20 pm'''
+
'''TO DO:'''
 +
* MP for N4 (A,B) and N5 (A,B)
 +
** Length check for N4 (A,B) and N5 (A,B)if successful proceed with digest and gel extract
 +
* Transform ligated construct : N1 = J23100C + I0462
 +
* retry E1A (may need to go back to verify that starting parts are working properly)
 +
* Transform Q04400 from the 2007 plates (I think the one we were using was from last year).  Same for I13033, unless the one we have is from 2007, then get the one from 2005 (2006 plate was bad in general)
-
Anam
+
== October 16, 2007 ==
-
*Did PD length check for Q04400 ACD, R0082 #1 & #2, J06501 AB, J13210 AB
+
Natalie (6:30 - 12:30)
-
**R0082: cut using X (linearize)
+
-
**Q04400: cut using X/S
+
-
**J13210: cut using X (linearize)
+
-
**J06501: cut using X/S
+
 +
From yesterday: E1A transformation unsuccessful; no colonies
-
''Recipe:''
+
Today:
-
*6 uL of ddH2O
+
* ligation of R0062 and E0433 (tube in freezer labelled "nat1")
-
*2 uL of plasmid
+
* ligation of I0462 and (R0062 + E0240) (tube in freezer labelled "nat2")
-
*1 uL of Buffer 2 (for all)
+
* Seema plated T9002 in DH5a
-
*0.5 uL of each enzyme
+
* stored half a clean gel in the fridge
-
 
+
* digested Q04400B and I13033 for length checking; stored gel in fridge for second-party verification
-
*Incubated for 40 min in incubator
+
** O = match, X = non-match
-
 
+
{| border=1
-
*''NOTE:'' NO BANDS SHOWED UP FOR Q04400 ACD
+
! Part Name !! Part !! Plasmid
-
 
+
-
{| border="1"
+
-
|+ Plasmid Length Check Results
+
|-
|-
-
! &nbsp; R0082 #1 !! R0082 #2 !! J13210 A !! J13210 B !! J06501 A !! J06501 B
+
| Q04400B || O || O
|-
|-
-
| '''2500'''-2000(very faint), '''1500'''-1000(bright) || '''2500'''-2000(very faint), '''1500'''-1000(bright) || 6000(faint), 5000-4000(bright), '''2500'''-2000(faint) || 6000(faint), 5000-4000(very faint), '''2500'''-2000(bright) || 3500-3000(very bright), 2000-1500(very, very faint) || 3500-3000(faint)
+
| I13033 || X? || O
|}
|}
 +
** may need to find part for I13033 using more finely grained ladder
 +
* made 3 extra AMP plates and stored the rest of the agar in a different beaker
 +
* prepared the plasmids from Calgary for transformation (pPCB, pCph8)
 +
** more like attempted! Will find out if it worked tomorrow... thanks, Patrick!
 +
* transformed parts into DH5a cells and plated:
 +
** R0062 + E0433
 +
** I0462 + (R0062 + E0240)
 +
** pPCB
 +
** pCph8
 +
*** note I spilled some ethanol on the pCph8 plate, which may adversely affect colony growth (ie. kill cells)
 +
To do tomorrow:
 +
* attend simulation meeting 12-1 in MSB caf (bring your own lunch)
 +
* retry E1A (may need to go back to verify that starting parts are working properly)
 +
* if transformations successful, do overnights of colonies; otherwise, try try again
 +
* gel extract, quantitate, ligate Q04400B
 +
* verify that I13033 is correct
 +
* update wiki page - project description and progress
-
'''Length Check Interpretation:'''
+
== Oct. 15th, 2007 ==
 +
yusuf, Irina
-
*R0082 #1 & #2:
+
* transformation of (J23100+S01640) + J06801 labelled as E1A
-
**1st band - P+P showed up and is correct because this was linearized
+
** used 4μl of ligated material from iGEM 2007 box
-
**2nd band - don't know what this is (it shouldn't have shown up theoretically). It cant be undigested plasmid because it travelled further than the P + P band (1st band).
+
** plate used AMP+Kan
-
*J13210 AB:
+
* MP of N2 (A,B,C,D) and length check
-
**1st band - May be undigested P + P
+
** Enzyme used Xba/Pst
-
**2nd band - Correct P + P length because this was linearized
+
** For all of N2 (A,B,C,D) the bands dont correspond to actual part and plasmid length
-
**3rd band - don't know what this is (it shouldn't have shown up theoretically).
+
** N2 (A,B,C,D) plasmid -> 3700bp from gel
-
*J06501 AB:
+
* LENGTH CHECK RESULTS:
-
**1st band - may be undigested plasmid
+
{| border=1
-
**2nd band (showed up only in A) - this is the correct size for the plasmid only but there's no band for the part.
+
|+ N2 Results
-
**NOTE: Part (and plasmid only for B) did not show up for both A & B
+
! Part Name !! Part !! Plasmid
 +
|-
 +
| A   ||  N/A  ||  x
 +
|-
 +
| B   ||  N/A   ||  x
 +
|-
 +
| C  ||  N/A  ||  x
 +
|-
 +
| D  ||  N/A  ||  x
 +
|}
-
+
* digest and length check for Q04400:
 +
** Enzyme used Xba/Pst
 +
** Not a match
-
* Made 2 tubes of MP o/n and put them in the incubator for S01640 B
+
* To do:
-
**We only have S01640 A in -80 freezer stock but since we have S01640 B in the output circuit and will be testing that, we can use that again in the backprop circuit if it works in the output circuit.
+
-
**MP one of them tomorrow and make the other one stock
+
-
* Did MP of B0034
+
** MP 0/n  for J06801A+(J23100+S01640)
-
** Found it in the igem box in the -80 freezer from last year's stock and Charles has said that we can use it.
+
** Charles figure something out for Q04400 and N2 because none of them was a match..
-
** It was labelled "B0034 (2005)" in last year's stock and so I have also labelled "B0034 (2005) MP" on the top of the tube with the MP and put today's date on the side. It is stored in the yellow IGEM box.
+
** Put the tips in the autoclave room
-
**Do PD length check for this.
+
-
'''OTHER THINGS TO DO AS WELL (Read full lab entry for all tasks):'''
+
== Oct. 14th, 2007 ==
-
*Transform I13033, E0433, S03140, J04500, J31003, and (J23100C + S01640B)D + B0015
+
Esther
-
*Make stock cells and LB(only one tube left...had to throw previous one because something was growing in there) ('''URGENT''')
+
-
== Sept. 28th, 2007 (Friday) ==
+
1. MP O/N of N2 A, B, C, D.
-
'''Time: 4:30pm - 10:00pm'''
+
2. Quantitation of
 +
*J06801A (Oct. 5th and Oct. 11th batch)
 +
*E0440A (2 μL of plasmid used)
 +
*Q04400 (2 μL of plasmid used)
 +
*Bands showed up for ALL of the above. J06801A was good. E0440A was sketchy, and Q04400 needs investigating. Check lab notebook for details, esp. on E04400A and Q04400A.
-
Yusuf, Fareeha
+
To be done by Adnan in the afternoon:
-
* Finished MP for Q04400 A, C, D
+
1. Ligation of J06801A and (J23100+S01640)
-
** Started doing length check for Q04400 A, C, D using enzymes '''Xba/Pst'''
+
*Both have been quantified and are in the enzyme box.  
-
** NO BANDS SHOWED UP FOR ALL THREE
+
-
** COULD BE DUE TO THE FACT THAT WE USED A '''REALLY OLD GEL SLICE FROM 2 WEEKS AGO''' BUT THE BANDS FOR THE LADDER SHOWED UP PERFECTLY..  NOTE POINT.. ASK SENIORS ABOUT IT
+
-
** THE COLONIES FROM WHERE Q04400 WAS TAKEN WAS '''IN THE INCUBATOR FOR 3 DAYS'''.. NOTE POINT.. ASK SENIORS HOW THAT WORKS
+
-
* LIGATED THE PART (J23100 C + S01640B)D WITH TERMINATOR B0015
+
2. PD and GE of
-
** INCUBATION TIME: 1HR 45 MIN AT R.T.P
+
*I13033
 +
*N1A
 +
*R0082 #1
 +
*I13504A
-
TO DO FOR TOMORROW:
+
To do (Monday):  
 +
# Transformation of J06801A+(J23100+S01640)
 +
# MP of N2 A,B,C,D
 +
# PD check and then PD, GE of N2
 +
# ligations from #2 (check flow chart)
-
* Transformation for the ligated part '''(J23100 C + S01640B)D WITH TERMINATOR B0015'''
+
== Oct. 13th, 2007 ==
-
* Try to resolve the part Q04400 controversy do a length check with another enzyme combination
+
-
** If that doesnt work then transform it again
+
-
* Length check and Gel extraction and digestion for R0082
+
Anam
-
** Just do the length check for R0082 and Q0040 together to save time
+
-
* I HAVE MADE A GEL FOR YOU IN THE FREEZER
+
*Retransformed N2 (New) into DH5a because the one from yesterday didn't grow
-
* CALL CHARLES UP TO COME UP WITH A PLAN AND TELL HIM TO GIVE US A "GO" SIGNAL WITH THE REST OF THE PARTS SO THAT WE CAN START TRANSFORMATION
+
**Use 5μL of DNA and transformed it on the same plate that was used yesterday (Charles said it should be okay since nothing grew on it).
 +
*PD, GE, and purification completed for J06801 (two tubes) cut using E/X. --> ligate with already purified J23100 + S01640 (it was cut with E/S).
 +
**PD was done for E0433 and Q04400 cut using X/P but the right bands didn't show up (refer to lab notebook for more details).
 +
**'''We are out of PstI.''' There are tubes of R0082, I13033, and N1 in the enzyme box in the with S/P written on them. They don't have enzymes in them. They just have the corresponding plasmid, BSA, and buffers in them. And so, it can be frozen. We can digest them when we have PstI.
 +
== Oct. 12th, 2007 ==
 +
Yusuf, Faareha, Talal
-
== Sept. 27th, 2007 (Thursday) ==
+
* Transformation of N2 (New) into (AMP + KAN) plate.
 +
** It's in incubator.
-
'''Hours: 5 PM - 10 PM'''
+
* MP of Q04400 (A,B)and I13033.
-
Esther
+
* Length check for Q04400 (A,B).
-
*Neha
+
-
*Rafsan
+
-
Results of the MP O/N from yesterday:
+
* Length check for R0082 (A,B)
-
*R0082 A, B = successful growth
+
** Part length: 108 bp
-
*Q0440 A, B = no growth
+
** Plasmid length: 2079 bp
-
Currently under the assumption that the incorrect antibiotic was added to the O/N for Q0440; retrying once again with KAN resistance.
+
* LENGTH CHECK RESULTS:
 +
{| border=1
 +
! Part Name !! Part !! Plasmid
 +
|-
 +
| Q04400 A || O || O
 +
|-
 +
| Q04400 B || O || O
 +
|-
 +
| R0082 1  || &nbsp; ||              O  *DIGESTED WITH ONE ENZYME (SPE1)
 +
|-
 +
| R0082 2  || &nbsp;    ||          O
 +
|-
 +
| I13033 ||      X  ||        O
 +
|}
-
Note: The antibiotic resistance is written on the plate. Be sure to check it before making an O/N.
+
* '''YEAHHH WE HAVE GOT QO4400 NOW WE CAN START OUR WORK'''  
-
 
+
-
1. MP of R0082 A, B
+
-
* Standard procedures used
+
-
* Note: tubes A and B got mixed up, they are now labelled #1 and #2 arbitrarily for differentiation. Colonies of origin are unknown. Re: No longer know which tube came from which colony.
+
-
 
+
-
2. Quantitation of (J23100 c + S01640B)D (now referred to in this entry as "insert")
+
-
*Brightness appeared intermediate between fifth band of the 1uL of HindIII ladder and 2uL of HindIII ladder. The lower concentration was used for sake of effectiveness.
+
-
*Chart read 47ng/ug, therefore [4.7ng/uL]
+
-
 
+
-
3. O/N of Q0440
+
-
*Colony A: may have had insufficient bacteria. Tried an O/N regardless.
+
-
*Colony B: insufficient bacteria. Did not try an O/N.
+
-
*Colonies C and D: O/N was done with these two, one to replace B and the other in case A was insufficient.
+
-
**Note: You can allow bacteria to regrow at room temperature or incubator if necessary.
+
-
 
+
-
Tomorrow:
+
-
*Plates must be made (shift started at 5 today, could not autoclave so did not make plates)
+
-
*LB must also be made
+
-
*Ligate (J2300+S01640) from today with B0015 from yesterday. The former is the insert, the latter is the plasmid. Recipe is in the lab notebook.
+
-
*If enough time, transform the above into a plate.
+
-
 
+
-
== Sept. 26th, 2007 (Wednesday) ==
+
-
 
+
-
'''Start Time: 3:30 pm - 9:00 pm'''
+
-
 
+
-
Yusuf
+
-
 
+
-
* Mini prep o/n from Sept. 24th 2007 is worthless since no body showed up to take it out and finish the MINI PREP. I was still counting on our prior schedule for September.
+
-
* SAME Mini prep o/n of '''(Q04400 -> A & B) & (R0082 -> A & B)'''
+
-
* Doing a digest to do a gel extraction for parts:
+
-
      '''(J23100C + S01640B)D'''
+
-
** For detailed info plz check the lab notebook.
+
-
 
+
-
* Finished quantitation for B0015
+
-
** Concentration written down in lab notebook and on the eppendorf tube as well.
+
-
 
+
-
TO DO FOR TOMORROW:
+
-
* Quantitation for  '''(J23100C + S01640B)D'''
+
-
* LIGATION FOR '' '''(J23100C + S01640B)D'''''  WITH '''''B0015'''''
+
-
** Esther make sure you do the calculation to figure out how much you are going to need properly. If not sure ask Seema or any one of us.
+
-
* MAKE SURE YOU FINISH THE MINI PREP O/N WHICH IS IN THE INCUBATOR
+
-
* MAKING PLATES, make sure you do it before the wet cycle which is at 3pm exact:
+
-
** 4 AMP + KAN
+
-
** 4 AMP
+
-
** 2 AMP + TET
+
-
** 2 KAN
+
-
* Ask Charles about what to do for the new parts that we are transforming and mini prepping.
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
== Sept. 24th, 2007 (Monday) ==
+
-
 
+
-
'''Start Time: 3:30 pm - 6 pm'''
+
-
 
+
-
Yusuf, Irina
+
-
* Mini Prep of J06501 A, J06501 B, J13210 A, J13210 B
+
* Enzyme activity
-
* Mini Prep o/n of Q04400 & (R0082 -> A & B)
+
** Recipe used:
-
* Made 4 AMP plates and 4 AMP+CAM plates
+
*** Enzyme: 0.25 μL
 +
*** Plasmid (J04500): 1 μL
 +
*** BSA: 0.5 μL
 +
*** Buffer: 0.5 μL
 +
*** Distilled water: 3.75 μL
 +
**RESULTS IN LAB NOTEBOOK: RESULTS PRETTY GOOD FOR ALL OF THEM
TO DO:
TO DO:
-
* Plasmid digest of (J23100C + S01640B)D
+
* LIGATE R0082 WITH E0240 A
-
* Then quantitation of (J23100C + S01640B)D and B0015
+
** TRANSFORM THE LIGATED CONSTRUCT
-
* Ligation of (J23100C + S01640B)D and B0015
+
 +
* MP O/N FOR N2
 +
* DIGEST, GEL EXTRACT, PURIFICATION OF Q04400
 +
* RECHECK LENGTH CHECK FOR I13033 BECAUSE THE PLASMID SHOWS UP BUT THE PART DOESNT
 +
* WE HAVE A LOT OF AGAR GELS IN THE FREEZER USE THEM
-
== Sept. 21st, 2007 (Friday) ==
+
== Oct. 11th, 2007 ==
 +
Esther, Neha
-
'''Start Time: 3:00 pm'''
+
1. MP O/N of
 +
*Q04400
 +
**6 tubes of colony A
 +
**6 tubes of colony B
 +
*I13033
 +
**6 tubes of colony A
 +
*Each tube contained 1mL of LB
-
Yusuf
+
2. PD of E0240A and Q04400B (old MP), along with enzyme checks (see lab notebook)
-
* Mini Prep o/n for '''(J23100 C + S01640 B) D'''.
+
3. Ligation of N1A and S01003. (N1A + S01003 = N2)
-
** REASONING: Seema suggested that instead of us having the RFP inside of I13507 and since we already checked by PLASMID LENGTH checks and fluorometry that this colony is successful, we could remove it since it sort of staying there is redundant. So all our previous work is sort of redundant as well.
+
-
THINGS TO DO FOR TOMMOROW: (ANAM, ESTHER)
+
4. MP of J06801 A&B
-
* Do mini prep for '''(J23100 C + S01640 B) D'''
+
To do:
-
* Plasmid digest for '''(J23100 C + S01640 B) D''' and '''B0015''' which is the terminator in MP form that Seema supplied us with.
+
-
*Make the desired plates that we will be needing and we have to confirm with Charles of which we are going to make. The Broth and Agar has been autoclaved and will be on the bench.
+
-
* Gel Extract and ligate both '''(J23100 C + S01640 B) D''' and '''B0015'''
+
-
* Possibly do transformations for the new construct that DAN gave us but have to confirm with Charles
+
-
* Tidy up notebook and ABBREVIATIONS and also the iGEM boxes in the -20c freezer.
+
 +
1. Recheck the relative efficiency of all enzymes in the box.
 +
2. Sample PD for R0082.
 +
*if good, takes steps to ligate with E0240A
 +
*if not good, troubleshoot - remake the MP with different colonies, etc.
-
== Sept. 19th, 2007 (Wednesday) ==
+
3. MP of Q04400 (A and B) and I13033.
-
'''Start Time: 3:30pm - End Time: 9:00pm'''
+
4. Transformation of N2
 +
== Oct. 10th, 2007 ==
Yusuf
Yusuf
-
* Plasmid Digest of (*L-D + I13507A)A using 22ul of mini prep.
+
* Transformation of Q04400 in DH5a from DNA of 2006
-
** There are about 20ul of mini prep left the yellow labeled igem box.
+
** Its in the incubator and needs to be MP o/n tomorrow
-
* Enzymes used was S/P.
+
-
* [['''PROBLEM:''']] The band that was formed was very thick so it incorporated up to 3 bands in the 1kb ladder.
+
* Quantitation of N1 and S01003 using previously digested parts
-
* The ladders that it incorporated was 5, 6, & 7 even though our expected bp was supposed to be 3093bp.
+
** '''Whoever comes in tomorrow plz check if the parts match up. everything is in the notebook and also do the calculation to find out the concentration of both constructs if they match up to what is expected'''
-
* We have to keep that in mind for the future if any errors arise.
+
-
* The gel has been cut and have been purified and the purified DNA has been put in to the yellow labeled iGem box.
+
 +
* Length check of the following:
 +
** E1B # 1 (A,B)
 +
** E1B # 2 (A,B)
 +
** R0082 (A,B)
 +
** I13504 (A,B)
 +
*** For all these length checks enzyme combination Xba/Pst was used
 +
*Length Check:
 +
{| border=1
 +
! Part Name !! Part !! Plasmid
 +
|-
 +
| E1B#1 A || O ||    O
 +
|-
 +
| E1B#1 B || O ||    O
 +
|-
 +
| E1B#2 A || O ||    O
 +
|-
 +
| E1B#2 B || O ||    O
 +
|-
 +
| I13504 A || O ||    O
 +
|-
 +
| I13504 B || x ||    x
 +
|-
 +
| R0082 A || x ||    O
 +
|-
 +
| R0082 B || x ||    O
 +
|}
 +
* Mp o/n of J06801
 +
* I13033: plate couldnt be found
 +
* Made:
 +
** 3 AMP+KAN
 +
** 3 KAN
-
== Sept. 17, 2007 ==
+
TO DO:
 +
* MP o/n of Q04400
 +
* Ligation of N1 + S01003 using previously digested parts
 +
** Quantitation has already been done and the tubes for these are in the iGem 2007 box
 +
* Proceed with ligating OmpR promoter with GFP: R0082 + E0240. (Use I13504 if you think it's necessary)
 +
* Test S/P/E/X enzymes (all of them) to determine relative activity and record the order.  Use one of the not commonly used plasmids
 +
* MP of J06801 A&B
 +
* Ligate if N1 and S01003 is correct
-
Anam
+
== Oct. 9, 2007 ==
-
'''(Outside lab stuff):'''
+
* Digest, GE, purify, quantitate:
 +
** I13033 A, S/P - Bands at: ~3500, ~3000 (cut out), 2000-1500
 +
*** [I13033] = 0 ng/μL
 +
** J06801 A, E/X - Bands at: ~6000 (cut out)
 +
*** [J06801] = 0 ng/μL
 +
** Q04400 C, X/P - Bands at: none
 +
* Mini-prep E1b #1 AB, E1b #2 AB (#1 and #2 denote plate numbers)
 +
* length check (O = match, X = non-match):
-
Regarding testing R0011 + F1610A, the following is the basic experimental procedure:
+
{| border=1
 +
! Part Name !! Part !! Plasmid
 +
|-
 +
| N2 A || X || O
 +
|-
 +
| N2 B || X || O
 +
|-
 +
| N2 C || X || O
 +
|-
 +
| N2 D || X || O
 +
|-
 +
|}
-
*Transform complete input circuit (R0011 + F1610) into dh5az-1 (done...its in the fridge...length checks have also been done for this)
+
* For N2, the part length corresponded to ~1000 bp, which is the length of both N1 and S01003.
-
*Transform complete output circuit (J23100 + S01640 + I13507 + S01003) into dh5a or dh5az-1
+
* Hold off on length checking R0062 (may or may not happen)
-
*Mix them up and test for cfp production
+
-
 
+
-
Theory:
+
-
 
+
-
*LuxR genes on output circuit produce luxR protein
+
-
*HSL (signal molecule) is produced from luxI gene in the input circuit and pushed out of the cell
+
-
*HSL diffuses, binds on to luxI protein and forms complex
+
-
*Activates the promoter (R0062) on S01003 in the output circuit and therefore, activates the production of cfp
+
'''To Do:'''
'''To Do:'''
-
*TO ALL F/T: familiarize yourself with the theoretical aspect of the updated project
+
* Length check of E1b #1 AB, E1b #2 AB
-
*PD, GE, and gel purification (*L-D + I13507A)A (the one that passed the fluorometer test...it has already been mini-prepped)
+
* Make plates when you need them. (We have 2 large agar flasks, and if you do melt one, maybe split it into 2 flasks)
-
** If you don't have time to purify the gel slices, store them in the -20 freezer and alert the next person who is coming into the lab.
+
* Proceed with ligating OmpR promoter with GFP: R0082 + E0240. (Use I13504 if you think it's necessary)
-
*Quantitate and ligate the S01003(any colony) and (*L-D + I13507A)A together and transform it (dh5a or dh5az-1..it doesnt matter for this circuit but personally i would do it in dh5a just so that when we converse about it, its easy to know which circuit we are referring to [since R0011+F1610A HAVE to be in the dh5az-1])
+
* Test S/P/E/X enzymes (all of them) to determine relative activity and record the order. Use one of the not commonly used plasmids
-
*PD length check for the complete output circuit after it is transformed (J23100 + S01640 + I13507 + S01003)
+
* Re-ligate N1 + S01003 using previously digested parts (make sure to quantitate again)
-
*Research excitation and emission wavelengths for cfp
+
* Transform Q04400 into DH5a
-
*Test for cfp production
+
* Make o/n of J06801 A, I13033 A (4 x 2.5 mL each) - this is to address the low DNA concentrations; we will combine them into 2 super MPs (the MP kit can only handle a max of 5 mL of o/n per tube).  We will then combine them in the gel extractions stage
-
 
+
== Oct. 8, 2007 ==
-
== Sept. 16, 2007 ==
+
-
 
+
-
Esther
+
-
 
+
-
*PD done for S01003 A, B, C, D
+
-
 
+
-
*42 uL of Plasmid
+
-
*6 uL of BSA
+
-
*6 uL of Buffer #2
+
-
*3 uL of XbaI
+
-
*3 uL of PstI
+
-
*3 uL Loading dye
+
-
*63 uL Total
+
-
 
+
-
Note on Enzymes: Enzymes are generally not consumed by the reaction. This means that you can digest the amounts of MP used in this lab with 1 uL of enzyme, but you will have to increase the digest time. So the short of it is to try to conserve enzyme when you can by digesting longer. Only increase your quantity of enzyme if you absolutely do not have the time to wait around.
+
-
 
+
-
Results:
+
-
 
+
-
colonies A, B:
+
-
* low yield
+
-
* 3 bands: 2000, 1500, 1000
+
-
 
+
-
colonies C, D:
+
-
* high yield
+
-
* 2 bands: 1500, 1000
+
-
 
+
-
Gel Extraction:
+
-
 
+
-
Weights
+
-
 
+
-
*A: 0.22g
+
-
*B: 0.23g
+
-
*C: 0.35g
+
-
*D: 0.31g
+
-
 
+
-
Buffer:
+
-
 
+
-
*A: 660uL
+
-
*B: 690uL
+
-
*C: 1050uL
+
-
*D: 930uL
+
-
 
+
-
* 30uL of TE buffer was used.
+
-
* extra allotment of TE buffer has been left out in an eppendorf tube holder. Please use it for the next gel purification.
+
-
 
+
-
== Sept. 15, 2007 ==
+
-
 
+
-
Anam
+
-
 
+
-
*Tested for rfp fluorescence using the fluorometer for J23100 + S01640 + I13507
+
-
*Only (*L-D + I13507A)A (the red colored ones) were produced rfp
+
-
 
+
-
'''To do:'''
+
-
*PD, GE, and gel purification of S01003 will be done on Sunday by whoever comes in
+
-
 
+
-
== Sept. 14, 2007 ==
+
-
 
+
-
Esther
+
-
 
+
-
Note: (J23100 + S01640)= *L
+
-
 
+
-
3 M/Ps was done with material from yesterday:
+
-
*1) (*L-B + I13507B)A x 2
+
-
*2) (*L-C + I13507C)A x 2
+
-
*3) (*L-D + I13507A)A x 2 (pink cells)
+
-
 
+
-
3 Freezer stocks made with material from yesterday:
+
-
*1) (*L-B + I13507B)A x 2
+
-
*2) (*L-C + I13507C)A x 2
+
-
*3) (*L-D + I13507A)A x 2 (pink cells)
+
-
 
+
-
Standard procedure was used for freezer stocks.
+
-
 
+
-
3 MPs:
+
-
*A standard MP was done.
+
-
 
+
-
Plasmid Digest:
+
-
*A sample digest was run for all the MP material.
+
-
 
+
-
*2 uL plasmid
+
-
*1 uL BSA
+
-
*1 uL buffer #2
+
-
*0.5 uL PstI
+
-
*0.5 uL XbaI
+
-
*5 uL ddH20
+
-
*3 uL 1 kb ladder
+
-
*2 uL loading dye
+
-
 
+
-
Results: All results were identical.
+
-
 
+
-
3 bands:
+
-
*1) 4000-'''3500'''
+
-
*2) 2500-'''2000'''
+
-
*3) '''2000'''-1500
+
-
 
+
-
Additional Note:
+
-
*MP O/N done for:
+
-
**1) (*L-B + I13507B)A x 2 (possibility of incorrect colony for first O/N. Second O/N done, labelled identically, but with "#2" written in for differentiation).
+
-
**2) (*L-C + I13507C)
+
-
**3) (*L-D + I13507A)
+
-
 
+
-
== Sept. 13, 2007 ==
+
-
 
+
-
Esther
+
-
 
+
-
*Six test tubes of MP O/N were used from yesterday:
+
-
**1) (*L-B + I13507B)A x 2
+
-
**2) (*L-C + I13507C)A x 2
+
-
**3) (*L-D + I13507A)A x 2
+
-
 
+
-
*Note: (J23100 + S01640)= *L
+
-
 
+
-
*Today was scheduled to be a fluorometer test for the above items, but there was some question of validity in terms of past data. To be re-examined.
+
-
 
+
-
*Therefore: All six tubes were left in the -20 freezer in eppendorf tubes after aspirating the supernatent.
+
-
 
+
-
== Sept. 12, 2007 ==
+
Yusuf
Yusuf
-
* MP o/n of the following:
+
* Mini prep o/n of the following:
 +
** E1B# 1 -> A&B
 +
** E1B# 2 -> A&B
-
** (*L-B + I13507 B ) '''A''' AMP RES = 2 copies
+
* Mini prep of the following:
-
** (*L-C + I13507 C ) '''A''' AMP RES = 2 copies
+
** N2 -> A,B,C,D
-
** (*L-D + I13507 A ) '''A''' AMP RES = 2 copies
+
** R0062 -> C,D,E,F
 +
* The above mini preps kept in the igem box labelled with green sticker "DNA from plates"
 +
TO DO:
 +
* Make
 +
** 5 AMP plates
 +
** 5 KAN Plates
 +
** 3 AMP+KAN Plates
-
== Sept 7th, 2007 ==
+
* Mini prep of:(in incubator)
 +
** E1B# 1 -> A&B
 +
** E1B# 2 -> A&B
-
=== Morning Session ===
+
* Length Check for:
 +
** E1B# 1 -> A&B
 +
** E1B# 2 -> A&B
 +
** N2 -> A,B,C,D
 +
** R0062 -> C,D,E,F
-
Yusuf, Neha
+
* Plasmid Digest and gel extract of the above constructs
-
Start Time: 11:15am - 2:00pm
+
== Oct. 7, 2007 ==
-
* MP of the following ligated parts:
+
Esther, Conrad, Adnan
-
** (*L-B + I13507 B ) '''A''' AMP RES
+
Done:
-
** (*L-B + I13507 B ) '''B''' AMP RES
+
* Transform E1b - Done with AMP plates. Two plates of this in the incubator. One may have been unsuccessful.
 +
* MP I13033A and J06801A  (two each; also freezer stocks of these, one each), MP of I13504 A, B.
 +
**'''Label may have been switched between the two parts. Run a sample PD to check before proceeding.'''  
 +
***The two that may have been switched are I13033A and J06801A - the ones that have the resistances written on them. The other ones were done later and there should be no problems.
 +
* MP o/n for N2 (4 colonies: A,B,C,D) and R0062 (colonies: C, D, E, F)
-
** (*L-C + I13507 C ) '''A''' AMP RES
+
To Do:
-
** (*L-C + I13507 C ) '''B''' AMP RES
+
*MP O/N of E1b
 +
*MP of N2 (A,B,C,D) and R0062 (C,D,E,F)
 +
* Digest, GE, purify, quantitate:
 +
** I13033 A, S/P
 +
** J06801 A, E/X
 +
** Q04400 C, X/P (assuming R0011 was cut with S/P)
 +
* PD length check I13504 AB
-
** (*L-D + I13507 A ) '''A''' AMP RES      '''''PINK COLORED CELLS'''''
+
Note: We are out of EtBr. Check with Seema.
-
** (*L-D + I13507 A ) '''B''' AMP RES      '''''PINK COLORED CELLS'''''
+
-
** (R0011 + F1610 A) '''A''' AMP RES
+
== Oct. 6, 2007 ==
-
** (R0011 + F1610 A) '''B''' AMP RES
+
-
** (R0011 + F1610 A) '''C''' AMP RES
+
-
** (R0011 + F1610 A) '''D''' AMP RES
+
 +
Anam, Tara, Jovan
 +
*Finished gel purification of J06801, J31003, B0034, E0433, I13033
 +
*MP for Q04400CD, R0062AB
 +
*PD length check for Q04400 CD, R0062AB
-
=== Evening Session ===
+
{| border=1
 +
! Part Name !! Part !! Plasmid
 +
|-
 +
| R0062 A || X || X
 +
|-
 +
| R0062 B || X || X
 +
|-
 +
| Q04400 C || O || O
 +
|-
 +
| Q04400 D || X || X
 +
|-
 +
|}
 +
 +
* Quantitation of J06801, J31003, B0034, E0433, I13033
 +
**Nothing showed up for J06801, I13033
 +
**[J31003] = 2.2 ng/μL
 +
**[B0034] = 9.4 ng/μL
 +
**[E0433] = 13.5 ng/μL
 +
 +
*Ligated B0034 + J31003 = E1b (refer to diagram posted up in wet lab area)
 +
 +
* Transformed N1A + S01003D = N2 and its in incubator.
 +
 +
*MP o/n for I13033A (2 copies), J06801A (2 copies), I13504 AB in incubator. (2 copies should be combined into 1 Eppendorf tube each; should end up with 1 new miniprep of I13033A and J06801A.  If these MPs are successful, remove/store the old ones)
-
Yusuf
 
-
 
-
* After MP I did PD length check for the above MPs. Enzymes X/P was used.
 
-
* After talking to Anam, we decided that the bands shown on the gel was acceptable, so we decided to go for MP o/n for the following:
 
-
 
-
** (*L-B + I13507 B ) '''A''' AMP RES
 
-
** (*L-B + I13507 B ) '''B''' AMP RES
 
-
 
-
** (*L-C + I13507 C ) '''A''' AMP RES
 
-
** (*L-C + I13507 C ) '''B''' AMP RES
 
-
 
-
** (*L-D + I13507 A ) '''A''' AMP RES      '''''PINK COLORED CELLS'''''
 
-
** (*L-D + I13507 A ) '''B''' AMP RES      '''''PINK COLORED CELLS'''''
 
-
 
-
* I have decided to do two MPs of each: For stock and for gel fluorescence.
 
-
 
-
 
-
 
-
== Sept 6, 2007 ==
 
-
 
-
Anam
 
-
 
-
Start time: 5pm
 
-
 
-
* 4 MP o/n for R0011 + F1610A (ABCD)
 
-
* 2 MP o/n per (J23100C + S01640B) + I13507 plate except for (J23100C + S01640B)A + I13507A because very tiny specks of colonies grew on the sides but the majority of the plate was just fuzzy looking (the plate looked cloudy). One other plate had some fuzziness (cloudy looking agar) in the middle but colonies grew on the sides and looked red (rfp production cuz the circuit works or just some mutation?? stay tuned to find out more).
 
-
** Possible problem with transformation yesterday: Had to throw away a LB tube because something was growing in it at the bottom. We used that tube for the first two transformations protocols that we did. Possibly a hand or something went over the tube when it was open and stuff went in and grew in the LB. The other ligations were incubated using LB from the other tube where (thankfully) nothing grew. This is a possible source of error. Hopefully the PD length check will reveal more.
 
-
 
-
'''NOTE:''' The colonies don't need to look red to the naked eye to know they are working. Seema has used some rfp (I13507) for her own work from a colony that is one of the ones we used and it worked well for her. She also indicated that her colonies didn't to look red, but the rfp showed up nice and bright when tested with the flourometer. So testing will also be performed on the colonies that pass the length check but don't look red to the naked eye.
 
'''TO DO:'''
'''TO DO:'''
-
* PD length check for all so that testing can be done on Saturday.
+
* Transform E1b
-
* After PD length check, make 3 mp o/n from 3 colonies that passed the length check (if those many pass...if there are lesser, just make 1 o/n per colony that passed) and the dilution process can begin on Saturday.
+
* MP I13033A, and J06801A (and make stock if there isn't any in the -80 freezer of this)
 +
** Don't do PD length check on these because it has already been done before and has passed it.
 +
* Digest, GE, purify, quantitate:
 +
** I13033 A, S/P
 +
** J06801 A, E/X
 +
** Q04400 C, X/P (assuming R0011 was cut with S/P)
 +
* MP and PD length check I13504 AB
 +
* MP o/n for N2 (4 colonies: A,B,C,D)
-
== Sept 5, 2007 ==
+
== Oct. 5, 2007 ==
-
=== Evening Session ===
+
Yusuf, Maria, Talal, Faareha
-
Start time: 5 pm
+
'''SUMMARY:'''
-
Anam, Dan
+
* Miniprep: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
 +
* Length Check:
 +
{| border=1
 +
! Part Name !! Part !! Plasmid
 +
|-
 +
| B0034 A || O || O
 +
|-
 +
| B0034 B || O || O
 +
|-
 +
| J06801 A || O || O
 +
|-
 +
| J06801 B || O || O
 +
|-
 +
| J31005 A || O || O
 +
|-
 +
| J31005 B || O || O
 +
|-
 +
| Q04400 A || O || O
 +
|-
 +
| Q04400 B || O || O
 +
|}
-
* Ligated R0011 + F1610
+
* Made competent cells
-
* Transformed ligated parts from yesterday and today
+
* Ligate N1 A + S01003 D = N2 (overnight)
-
** All, except one, were transformed in DH5a
+
* Made o/n of R0062 (2007) AB, Q04400 (2005) with high [IPTG] (pSB2k3 is high copy number in LacI- environment)
-
** R0011 + F1610 was transformed in DH5a-z1
+
* Transformed I13504 (2007)
 +
* Gel Extract:
 +
** B0034 A, S/P
 +
** E0433 A, X/P
 +
** I13033 A, S/P
 +
** J06801 A, E/X
 +
** J31003 A, X/P
'''TO DO:'''
'''TO DO:'''
-
* MP o/n for all transformed parts
 
-
== Sept 4, 2007 ==
+
* B0015 (2005) didn't transform, try again
 +
* Complete gel extract procedure
 +
* Ligate:
 +
** B0034 A + J31003 A
 +
** I13033 A + E0433 A
 +
** (J23100 + S01640) D + J06801 A
-
=== Morning Session ===
+
== Oct. 4, 2007 ==
-
Yusuf, Elliott
+
Esther, Neha, Rafsan
-
Start Time: 11:00am
+
'''SUMMARY:'''
-
* Making of stock cells:
+
* Found alternate to I1303: ligate terminator (B0015) and Lux pR promoter (R0062) separately
-
** one copy of F1610 (A)
+
* Transformed R0062 (2007), B0015 (2005)
-
** Two copies of - J23100 (C) + S0140 (B): All A, B, C, D
+
* Made o/n of DH5a, DH5a-z1, J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
-
*** Overall 8 copies
+
* Digest and Gel Extract of N1 A with enzymes S/P
-
 
+
* Quantitation: N1 = 16.8 ng/μL, S01003 (2007) = 4.1 ng/μL
-
* Made Plates:
+
-
** 7 AMP Plates
+
-
** 3 AMP and KAN Plates
+
-
 
+
-
* Sent pippette tips for autoclaving
+
-
 
+
-
* Quantitation for the following GEL EXTRACTS:
+
-
** J23100 (C) + S0140 (B): All A, B, C, D
+
-
** I13507: A, B, C
+
-
 
+
-
=== Evening Session ===
+
-
 
+
-
Anam, Dan, Maria
+
-
 
+
-
Start time: 5:30 pm
+
-
 
+
-
* Ligation of (J23100 C + S01640 B) + I13507
+
-
** (J23100 C + S01640 B) is labelled as *L - (colony it was picked from)
+
-
* Quantitation for F1610 A
+
'''TO DO:'''
'''TO DO:'''
-
* Ligate R0011 to F1610
 
-
* Tranform all the ligated parts
 
-
* PD, GE, and Gel purification for S01003
 
-
* Determine plan for testing input and output circuits and building other circuits (back prop)
 
-
* Check if we have DH5a-z1 cells and whether we need to make more (the input circuit needs to be transformed in DH5a-z1)
 
-
== Sept 3, 2007 ==
+
* Make o/n: R0062 (2007), B0015 (2005)
 +
* Miniprep and Check Lengths: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
 +
* Ligate N1 and S01003 and transform in DH5a
 +
* Make competent cells
-
Anam, Yusuf
+
== Oct. 3, 2007 ==
-
Start time: 12:30 pm
+
Yusuf
-
* 1 MP o/n of F1610 A for stock, 1 MP o/n for J23100C + S01640 B (ABCD) (from any colony, they all passed PD length check) for stock
+
'''SUMMARY:'''
-
* Use that gel for PD, GE, and Gel purification of I13507 (whichever colonies because all four worked), J23100C + S01640B (ABCD), and F1610 A
+
-
To do tomorrow:
+
* Transformed: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
-
 
+
* Minipreped I130033 AB, E0433 AB, S03140 AB, J04500 AB, J31003 AB, N1 AB
-
* Make AMP plates
+
* Made o/n of DH5a and DH5a-z1 (from July 16 plate - ask Seema if this is ok)
-
* Quantitate J23100 C + S01640 B (ABCD), I13507 ABC, and F1610 A
+
* Length Check (O = match, X = does not match):
-
* Ligate (J23100 C + S01640 B) + I13507 and R0011 + F1610
+
{| border=1
-
* Transformation of the ligated parts (if time permits)
+
! Part Name !! Part !! Plasmid
-
 
+
-
== Sept 2, 2007 ==
+
-
 
+
-
Anam
+
-
 
+
-
Start time: 9:30 am
+
-
 
+
-
* MP of J23100 C + S01640 B (ABCD)
+
-
* PD length checks for F1610 AB (from 2006 plates), J23100 C + S01640 B (ABCD)
+
-
** F1610 AB cut using X/P
+
-
** J23100 C + S01640 B (ABCD) cut using X/P
+
-
 
+
-
{| border="1"
+
-
|+ '''Plasmid Length Check Results'''
+
|-
|-
-
! &nbsp; !! F1610 !! J23100 C + S01640 B
+
| E0433 A || O || O
|-
|-
-
! A
+
| E0433 B || O || O
-
| Y || Y
+
|-
|-
-
! B
+
| I13033 A || X || O
-
| N || Y
+
|-
|-
-
! C
+
| I13033 B || X || O
-
| - || Y
+
|-
|-
-
! D
+
| J04500 A || O || O
-
| - || Y
+
|-
 +
| J04500 B || O || O
 +
|-
 +
| J31003 A || O || O
 +
|-
 +
| J31003 B || O || O
 +
|-
 +
| N1 A || O || O
 +
|-
 +
| N1 B || O || O
 +
|-
 +
| S03140 A || O || O
 +
|-
 +
| S03140 B || O || O
|}
|}
-
'''Legend:'''
+
'''TO DO:'''
-
Y - Yes
+
* Make 5 Kan plates
 +
* Make bottle of LB Broth
 +
* Make competent cells
 +
* Make o/n of J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
 +
* Ligate N1 + S01003
 +
* Find alternate to I13033
-
N - No
+
== Oct. 2, 2007 ==
-
'''To do list:'''
+
Natalie
-
* make AMP plates
+
'''SUMMARY:'''
-
* 1 MP o/n of F1610 A for stock, 1 MP o/n for J23100C + S01640 B (ABCD) (from any colony, they all passed PD length check) for stock
+
-
* make gel with big wells by taking the well maker with the big teeth (8 teeth) and use autoclave tape to join two of them
+
-
** Remember to leave spaces between wells when running gel for extraction
+
-
** Determine layout of gel before making the gel
+
-
* Use that gel for PD, GE, and Gel purification of I13507 (whichever colonies because all four worked), J23100C + S01640B (ABCD), and F1610 A.
+
-
** Put a total volume of 150-200μL in those big wells
+
-
== Sept 1, 2007 ==
+
* Made o/n (2 each) of: I130033 AB, E0433 AB, S03140 AB, J04500 AB, J31003 AB, N1 AB
 +
* NOTE: N1 = J23100 C + S01640 B + B0015
-
Anam
+
'''TO DO:'''
-
Start time: 6 pm
+
* Miniprep all above parts and length check
 +
* Transform:  
 +
** J06801 (2005) - it's in the source box (this is a larger composite part containing J06501)
 +
** B0034 - also in source box, if not, get it from Registry plates
 +
** Q04400 - re-transform using more DNA to see if we get more colonies and transform 2005/2006 version in the source box
 +
** J31005 - chloramphenicol resistance, just in case we need it
 +
* Continue cataloging and organization (Charles: I'll be dropping by to help out with that)
-
* MP F1610 AB
+
== Oct. 1, 2007 ==
-
* MP o/n J23100 C + S01640 B (AB)
+
-
** J23100 A + S01414 A and J23100 B + S01414 B didn't grow on their plates
+
-
== August 31, 2007 ==
+
'''Time: 3:45 PM - 8:30 PM'''
-
F/T: Anam
+
Yusuf, Irina
-
Start time: 8 am
+
Summary:
-
* Quantitated J23100 C, S01414 AB, & S01640 AB.
+
* made 5 KAN plates
-
* Ligated J23100 AB to S01414 AB (respectively), J23100 C to S01640 B, and J23100 D to S01640 A.
+
* made 2 AMP+KAN plates
 +
* made 300 mL LB
-
Start time: 5:30 pm
+
Tranformation of following parts:
 +
* I13033 -> KAN
 +
* E0433 -> KAN
 +
* S03140 -> AMP
 +
* J04500 -> KAN
 +
* J31003 -> AMP
 +
* (J23100C + S01640B)+ B0015 -> KAN
-
* Transformed J23100 A + S01414 A, J23100 B + S01414 B, J23100 C + S01640 B in DH5A cells.
+
Used 4μL of DNA for all transformations
-
** J23100 D + S01640 A was not transformed because there are only three AMP plates.
+
-
* MP o/n F1610 AB from the plate Seema gave us (part was from 2006 plate).
+
-
** Incubating o/n in shaker.
+
-
'''TO DO:'''
+
* tried to do DNA extraction for J06501 but somebody already extracted it (wasn't in the well)
-
* MP F1610 and do PD length check for it.
+
* couldn't find it in the iGEM DNA box as well !!!
-
* MP o/n the transformed ligated J23100 + insert (S01414 or S01640)
+
-
** How many colonies should we test from each plate?
+
-
* Make AMP plates
+
-
* Transform the other ligated J23100 D + SO1640 A
+
-
P/T: Rafsan
+
TO DO:
-
 
+
-
Start time: 6:30 pm
+
-
* Created stocks of parts to store in -80°C freezer.
+
* MP o/n for the above parts
-
 
+
-
PROCEDURE:
+
-
* Did MP o/n yesterday
+
-
* Added 500 μL of MP o/n solution in eppendorf tube
+
-
* Added 500 μL of Glycerol in eppendorf tube & mix well
+
-
* Filled 5 eppendorf tubes per part
+

Latest revision as of 04:01, 27 October 2007

Contents

< August | September | Current >

Parts Catalogue

October 24

  • ligate JI + N4 (AMP) into DH5a; this will be module D
  • ligate JI + RE (AMP) into DH5a; this will be module A
  • enzyme contamination test: cross-contamination detected in Xba and Spe tubes.

October 23

  • length check JI (J23100 + I0462): pass
  • quantitate JI, N4, Q04400B:
    • JI: 4.5 ng/uL
    • N4: 4.5 ng/uL
    • Q04400B: no bands seen
  • overnight prep of T9002(CDE), Q04400E
  • length check of E1A(AB): pass, but ghost bands present. Check for enzyme contamination.

October 20

(Anam)

  • miniprepped T9002(AB), E1A(AB)
  • quantitated Q04400B: no bands seen
  • length checks of T9002, N4, N5: all pass

October 19, 2007

Yusuf, Maria, Faareha, Talal

Start: 3pm - 12am

SUMMARY:

  • MP O/N for E1A and T9002
  • Transformation of J23100C and I0462 since no colonies grew
  • Quantitation for the following constructs (with results):
    • I13033 A - no band seen
    • I13504 with X/P - no band seen
    • N1A from Oct 4/07 with S/P - 13.5ng/μL
    • N1A from Oct 12/07 - 13.5ng/μL
    • R0082 - 5.5ng/μL
  • Digest
    • Q04400 B - primary digest with X/P wasn't successful. Only plasmid was seen. Second digest with E/S
      • If digest of Q04400 has been ok ed by charles proceed to Quantitation and ligation and transformation
    • Enzyme check for SpeI - all enzymes working

TO DO:

    • MP E1A and T9002
    • MP O/N for (J23100C + I0462)
    • Make AMP plates
    • After Charles approves it, continue to quantitation and ligation for Q04400B
    • Re quantitate the following I13033 and I13504 bcause no bands were seen when i did the quantitation
  • make competent cells, both DH5a and DH5a-z1 (Make about 12 each b/c we don't have that many tubes)
  • Charles will transform the pPCB and pCph8 plasmids

October 18th, 2007

Natalie

  • Rechecked Q04400B - everything is OK (verified by Seema and Esther).

Esther, Neha, Rafsan

1. MPs:

  • N4 (A,B) x2
  • N5 (A,B) x2
  • Stock cells of all of the above in the -80°C freezer
  • Some MP leftover - supernatent drained, put into -20°C freezer (if MP from today is poor, use these to redo the MP).

2. Transformation of E1b, N1 (new)

  • In the incubator.
  • Something is odd about these stock cells - they were taken from the -80°C freezer, iGEM box, labeled with "alpha" (presumably they are DH5a). Discuss at tomorrow's meeting.

3. PD of N4 A,B and N5 A,B - used X/P

4. Quantitation:

  • I13033
  • N1A
  • R0082 #1
  • I13504A

Note: The gel used was yesterday's.

Results:

Inconclusive. I have some doubts about the integrity of the wells in the gel - some of them were stuck together, some allowed loading dye to float out, etc. As a result, I am going to ask you redo the check for all of the above using a new gel.

To do:

1. MP O/N of E1b, N1 2. PD check: PD of N4 A,B and N5 A,B 3. Quantitate:

  • I13033
  • N1A
  • R0082 #1
  • I13504A

4. PD and GE of Q04400B - use the EXACT tube Natalie used yesterday - check the date (this is noted in the lab notebook).

October 17th, 2007

Yusuf (5:00pm - 10:00pm)

SUMMARY:

  • As optimistic as Natalie was, there is no way the parts Q04400B and I13033 match up with the bands seen in the gel. So no match once again.
  • Ligation of J23100C with I0462
    • the part has been named as N1 in the iGem Parts Excel sheet. Discard all other N1 and only keep the new N1
  • Mini prep O/N for N4 (A,B) and N5 (A,B)

TO DO:

  • MP for N4 (A,B) and N5 (A,B)
    • Length check for N4 (A,B) and N5 (A,B)if successful proceed with digest and gel extract
  • Transform ligated construct : N1 = J23100C + I0462
  • retry E1A (may need to go back to verify that starting parts are working properly)
  • Transform Q04400 from the 2007 plates (I think the one we were using was from last year). Same for I13033, unless the one we have is from 2007, then get the one from 2005 (2006 plate was bad in general)

October 16, 2007

Natalie (6:30 - 12:30)

From yesterday: E1A transformation unsuccessful; no colonies

Today:

  • ligation of R0062 and E0433 (tube in freezer labelled "nat1")
  • ligation of I0462 and (R0062 + E0240) (tube in freezer labelled "nat2")
  • Seema plated T9002 in DH5a
  • stored half a clean gel in the fridge
  • digested Q04400B and I13033 for length checking; stored gel in fridge for second-party verification
    • O = match, X = non-match
Part Name Part Plasmid
Q04400B O O
I13033 X? O
    • may need to find part for I13033 using more finely grained ladder
  • made 3 extra AMP plates and stored the rest of the agar in a different beaker
  • prepared the plasmids from Calgary for transformation (pPCB, pCph8)
    • more like attempted! Will find out if it worked tomorrow... thanks, Patrick!
  • transformed parts into DH5a cells and plated:
    • R0062 + E0433
    • I0462 + (R0062 + E0240)
    • pPCB
    • pCph8
      • note I spilled some ethanol on the pCph8 plate, which may adversely affect colony growth (ie. kill cells)

To do tomorrow:

  • attend simulation meeting 12-1 in MSB caf (bring your own lunch)
  • retry E1A (may need to go back to verify that starting parts are working properly)
  • if transformations successful, do overnights of colonies; otherwise, try try again
  • gel extract, quantitate, ligate Q04400B
  • verify that I13033 is correct
  • update wiki page - project description and progress

Oct. 15th, 2007

yusuf, Irina

  • transformation of (J23100+S01640) + J06801 labelled as E1A
    • used 4μl of ligated material from iGEM 2007 box
    • plate used AMP+Kan
  • MP of N2 (A,B,C,D) and length check
    • Enzyme used Xba/Pst
    • For all of N2 (A,B,C,D) the bands dont correspond to actual part and plasmid length
    • N2 (A,B,C,D) plasmid -> 3700bp from gel
  • LENGTH CHECK RESULTS:
N2 Results
Part Name Part Plasmid
A N/A x
B N/A x
C N/A x
D N/A x
  • digest and length check for Q04400:
    • Enzyme used Xba/Pst
    • Not a match
  • To do:
    • MP 0/n for J06801A+(J23100+S01640)
    • Charles figure something out for Q04400 and N2 because none of them was a match..
    • Put the tips in the autoclave room

Oct. 14th, 2007

Esther

1. MP O/N of N2 A, B, C, D.

2. Quantitation of

  • J06801A (Oct. 5th and Oct. 11th batch)
  • E0440A (2 μL of plasmid used)
  • Q04400 (2 μL of plasmid used)
  • Bands showed up for ALL of the above. J06801A was good. E0440A was sketchy, and Q04400 needs investigating. Check lab notebook for details, esp. on E04400A and Q04400A.

To be done by Adnan in the afternoon:

1. Ligation of J06801A and (J23100+S01640)

  • Both have been quantified and are in the enzyme box.

2. PD and GE of

  • I13033
  • N1A
  • R0082 #1
  • I13504A

To do (Monday):

  1. Transformation of J06801A+(J23100+S01640)
  2. MP of N2 A,B,C,D
  3. PD check and then PD, GE of N2
  4. ligations from #2 (check flow chart)

Oct. 13th, 2007

Anam

  • Retransformed N2 (New) into DH5a because the one from yesterday didn't grow
    • Use 5μL of DNA and transformed it on the same plate that was used yesterday (Charles said it should be okay since nothing grew on it).
  • PD, GE, and purification completed for J06801 (two tubes) cut using E/X. --> ligate with already purified J23100 + S01640 (it was cut with E/S).
    • PD was done for E0433 and Q04400 cut using X/P but the right bands didn't show up (refer to lab notebook for more details).
    • We are out of PstI. There are tubes of R0082, I13033, and N1 in the enzyme box in the with S/P written on them. They don't have enzymes in them. They just have the corresponding plasmid, BSA, and buffers in them. And so, it can be frozen. We can digest them when we have PstI.

Oct. 12th, 2007

Yusuf, Faareha, Talal

  • Transformation of N2 (New) into (AMP + KAN) plate.
    • It's in incubator.
  • MP of Q04400 (A,B)and I13033.
  • Length check for Q04400 (A,B).
  • Length check for R0082 (A,B)
    • Part length: 108 bp
    • Plasmid length: 2079 bp
  • LENGTH CHECK RESULTS:
Part Name Part Plasmid
Q04400 A O O
Q04400 B O O
R0082 1   O *DIGESTED WITH ONE ENZYME (SPE1)
R0082 2   O
I13033 X O
  • YEAHHH WE HAVE GOT QO4400 NOW WE CAN START OUR WORK
  • Enzyme activity
    • Recipe used:
      • Enzyme: 0.25 μL
      • Plasmid (J04500): 1 μL
      • BSA: 0.5 μL
      • Buffer: 0.5 μL
      • Distilled water: 3.75 μL
    • RESULTS IN LAB NOTEBOOK: RESULTS PRETTY GOOD FOR ALL OF THEM

TO DO:

  • LIGATE R0082 WITH E0240 A
    • TRANSFORM THE LIGATED CONSTRUCT
  • MP O/N FOR N2
  • DIGEST, GEL EXTRACT, PURIFICATION OF Q04400
  • RECHECK LENGTH CHECK FOR I13033 BECAUSE THE PLASMID SHOWS UP BUT THE PART DOESNT
  • WE HAVE A LOT OF AGAR GELS IN THE FREEZER USE THEM

Oct. 11th, 2007

Esther, Neha

1. MP O/N of

  • Q04400
    • 6 tubes of colony A
    • 6 tubes of colony B
  • I13033
    • 6 tubes of colony A
  • Each tube contained 1mL of LB

2. PD of E0240A and Q04400B (old MP), along with enzyme checks (see lab notebook)

3. Ligation of N1A and S01003. (N1A + S01003 = N2)

4. MP of J06801 A&B

To do:

1. Recheck the relative efficiency of all enzymes in the box.

2. Sample PD for R0082.

  • if good, takes steps to ligate with E0240A
  • if not good, troubleshoot - remake the MP with different colonies, etc.

3. MP of Q04400 (A and B) and I13033.

4. Transformation of N2

Oct. 10th, 2007

Yusuf

  • Transformation of Q04400 in DH5a from DNA of 2006
    • Its in the incubator and needs to be MP o/n tomorrow
  • Quantitation of N1 and S01003 using previously digested parts
    • Whoever comes in tomorrow plz check if the parts match up. everything is in the notebook and also do the calculation to find out the concentration of both constructs if they match up to what is expected
  • Length check of the following:
    • E1B # 1 (A,B)
    • E1B # 2 (A,B)
    • R0082 (A,B)
    • I13504 (A,B)
      • For all these length checks enzyme combination Xba/Pst was used
  • Length Check:
Part Name Part Plasmid
E1B#1 A O O
E1B#1 B O O
E1B#2 A O O
E1B#2 B O O
I13504 A O O
I13504 B x x
R0082 A x O
R0082 B x O
  • Mp o/n of J06801
  • I13033: plate couldnt be found
  • Made:
    • 3 AMP+KAN
    • 3 KAN

TO DO:

  • MP o/n of Q04400
  • Ligation of N1 + S01003 using previously digested parts
    • Quantitation has already been done and the tubes for these are in the iGem 2007 box
  • Proceed with ligating OmpR promoter with GFP: R0082 + E0240. (Use I13504 if you think it's necessary)
  • Test S/P/E/X enzymes (all of them) to determine relative activity and record the order. Use one of the not commonly used plasmids
  • MP of J06801 A&B
  • Ligate if N1 and S01003 is correct

Oct. 9, 2007

  • Digest, GE, purify, quantitate:
    • I13033 A, S/P - Bands at: ~3500, ~3000 (cut out), 2000-1500
      • [I13033] = 0 ng/μL
    • J06801 A, E/X - Bands at: ~6000 (cut out)
      • [J06801] = 0 ng/μL
    • Q04400 C, X/P - Bands at: none
  • Mini-prep E1b #1 AB, E1b #2 AB (#1 and #2 denote plate numbers)
  • length check (O = match, X = non-match):
Part Name Part Plasmid
N2 A X O
N2 B X O
N2 C X O
N2 D X O
  • For N2, the part length corresponded to ~1000 bp, which is the length of both N1 and S01003.
  • Hold off on length checking R0062 (may or may not happen)

To Do:

  • Length check of E1b #1 AB, E1b #2 AB
  • Make plates when you need them. (We have 2 large agar flasks, and if you do melt one, maybe split it into 2 flasks)
  • Proceed with ligating OmpR promoter with GFP: R0082 + E0240. (Use I13504 if you think it's necessary)
  • Test S/P/E/X enzymes (all of them) to determine relative activity and record the order. Use one of the not commonly used plasmids
  • Re-ligate N1 + S01003 using previously digested parts (make sure to quantitate again)
  • Transform Q04400 into DH5a
  • Make o/n of J06801 A, I13033 A (4 x 2.5 mL each) - this is to address the low DNA concentrations; we will combine them into 2 super MPs (the MP kit can only handle a max of 5 mL of o/n per tube). We will then combine them in the gel extractions stage

Oct. 8, 2007

Yusuf

  • Mini prep o/n of the following:
    • E1B# 1 -> A&B
    • E1B# 2 -> A&B
  • Mini prep of the following:
    • N2 -> A,B,C,D
    • R0062 -> C,D,E,F
  • The above mini preps kept in the igem box labelled with green sticker "DNA from plates"

TO DO:

  • Make
    • 5 AMP plates
    • 5 KAN Plates
    • 3 AMP+KAN Plates
  • Mini prep of:(in incubator)
    • E1B# 1 -> A&B
    • E1B# 2 -> A&B
  • Length Check for:
    • E1B# 1 -> A&B
    • E1B# 2 -> A&B
    • N2 -> A,B,C,D
    • R0062 -> C,D,E,F
  • Plasmid Digest and gel extract of the above constructs

Oct. 7, 2007

Esther, Conrad, Adnan

Done:

  • Transform E1b - Done with AMP plates. Two plates of this in the incubator. One may have been unsuccessful.
  • MP I13033A and J06801A (two each; also freezer stocks of these, one each), MP of I13504 A, B.
    • Label may have been switched between the two parts. Run a sample PD to check before proceeding.
      • The two that may have been switched are I13033A and J06801A - the ones that have the resistances written on them. The other ones were done later and there should be no problems.
  • MP o/n for N2 (4 colonies: A,B,C,D) and R0062 (colonies: C, D, E, F)

To Do:

  • MP O/N of E1b
  • MP of N2 (A,B,C,D) and R0062 (C,D,E,F)
  • Digest, GE, purify, quantitate:
    • I13033 A, S/P
    • J06801 A, E/X
    • Q04400 C, X/P (assuming R0011 was cut with S/P)
  • PD length check I13504 AB

Note: We are out of EtBr. Check with Seema.

Oct. 6, 2007

Anam, Tara, Jovan

  • Finished gel purification of J06801, J31003, B0034, E0433, I13033
  • MP for Q04400CD, R0062AB
  • PD length check for Q04400 CD, R0062AB
Part Name Part Plasmid
R0062 A X X
R0062 B X X
Q04400 C O O
Q04400 D X X
  • Quantitation of J06801, J31003, B0034, E0433, I13033
    • Nothing showed up for J06801, I13033
    • [J31003] = 2.2 ng/μL
    • [B0034] = 9.4 ng/μL
    • [E0433] = 13.5 ng/μL
  • Ligated B0034 + J31003 = E1b (refer to diagram posted up in wet lab area)
  • Transformed N1A + S01003D = N2 and its in incubator.
  • MP o/n for I13033A (2 copies), J06801A (2 copies), I13504 AB in incubator. (2 copies should be combined into 1 Eppendorf tube each; should end up with 1 new miniprep of I13033A and J06801A. If these MPs are successful, remove/store the old ones)


TO DO:

  • Transform E1b
  • MP I13033A, and J06801A (and make stock if there isn't any in the -80 freezer of this)
    • Don't do PD length check on these because it has already been done before and has passed it.
  • Digest, GE, purify, quantitate:
    • I13033 A, S/P
    • J06801 A, E/X
    • Q04400 C, X/P (assuming R0011 was cut with S/P)
  • MP and PD length check I13504 AB
  • MP o/n for N2 (4 colonies: A,B,C,D)

Oct. 5, 2007

Yusuf, Maria, Talal, Faareha

SUMMARY:

  • Miniprep: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
  • Length Check:
Part Name Part Plasmid
B0034 A O O
B0034 B O O
J06801 A O O
J06801 B O O
J31005 A O O
J31005 B O O
Q04400 A O O
Q04400 B O O
  • Made competent cells
  • Ligate N1 A + S01003 D = N2 (overnight)
  • Made o/n of R0062 (2007) AB, Q04400 (2005) with high [IPTG] (pSB2k3 is high copy number in LacI- environment)
  • Transformed I13504 (2007)
  • Gel Extract:
    • B0034 A, S/P
    • E0433 A, X/P
    • I13033 A, S/P
    • J06801 A, E/X
    • J31003 A, X/P

TO DO:

  • B0015 (2005) didn't transform, try again
  • Complete gel extract procedure
  • Ligate:
    • B0034 A + J31003 A
    • I13033 A + E0433 A
    • (J23100 + S01640) D + J06801 A

Oct. 4, 2007

Esther, Neha, Rafsan

SUMMARY:

  • Found alternate to I1303: ligate terminator (B0015) and Lux pR promoter (R0062) separately
  • Transformed R0062 (2007), B0015 (2005)
  • Made o/n of DH5a, DH5a-z1, J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
  • Digest and Gel Extract of N1 A with enzymes S/P
  • Quantitation: N1 = 16.8 ng/μL, S01003 (2007) = 4.1 ng/μL

TO DO:

  • Make o/n: R0062 (2007), B0015 (2005)
  • Miniprep and Check Lengths: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
  • Ligate N1 and S01003 and transform in DH5a
  • Make competent cells

Oct. 3, 2007

Yusuf

SUMMARY:

  • Transformed: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
  • Minipreped I130033 AB, E0433 AB, S03140 AB, J04500 AB, J31003 AB, N1 AB
  • Made o/n of DH5a and DH5a-z1 (from July 16 plate - ask Seema if this is ok)
  • Length Check (O = match, X = does not match):
Part Name Part Plasmid
E0433 A O O
E0433 B O O
I13033 A X O
I13033 B X O
J04500 A O O
J04500 B O O
J31003 A O O
J31003 B O O
N1 A O O
N1 B O O
S03140 A O O
S03140 B O O

TO DO:

  • Make 5 Kan plates
  • Make bottle of LB Broth
  • Make competent cells
  • Make o/n of J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
  • Ligate N1 + S01003
  • Find alternate to I13033

Oct. 2, 2007

Natalie

SUMMARY:

  • Made o/n (2 each) of: I130033 AB, E0433 AB, S03140 AB, J04500 AB, J31003 AB, N1 AB
  • NOTE: N1 = J23100 C + S01640 B + B0015

TO DO:

  • Miniprep all above parts and length check
  • Transform:
    • J06801 (2005) - it's in the source box (this is a larger composite part containing J06501)
    • B0034 - also in source box, if not, get it from Registry plates
    • Q04400 - re-transform using more DNA to see if we get more colonies and transform 2005/2006 version in the source box
    • J31005 - chloramphenicol resistance, just in case we need it
  • Continue cataloging and organization (Charles: I'll be dropping by to help out with that)

Oct. 1, 2007

Time: 3:45 PM - 8:30 PM

Yusuf, Irina

Summary:

  • made 5 KAN plates
  • made 2 AMP+KAN plates
  • made 300 mL LB

Tranformation of following parts:

  • I13033 -> KAN
  • E0433 -> KAN
  • S03140 -> AMP
  • J04500 -> KAN
  • J31003 -> AMP
  • (J23100C + S01640B)+ B0015 -> KAN

Used 4μL of DNA for all transformations

  • tried to do DNA extraction for J06501 but somebody already extracted it (wasn't in the well)
  • couldn't find it in the iGEM DNA box as well !!!

TO DO:

  • MP o/n for the above parts
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