Toronto/Lab Notebook

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== August 31, 2007 ==
+
{{TOCright}}
-
F/T: Anam
+
<center>
 +
<font size=4>< [[Toronto/Lab Notebook/August|August]] | [[Toronto/Lab Notebook/September|September]] | [[Toronto/Lab Notebook|Current]] ></font>
 +
</center>
-
Start time: 8 am
+
[[Toronto/PartsCatalogue|Parts Catalogue]]
-
* Quantitated J23100 C, S01414 AB, & S01640 AB.
+
== October 24 ==
-
* Ligated J23100 AB to S01414 AB (respectively), J23100 C to S01640 B, and J23100 D to S01640 A.
+
* 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.
-
Start time: 5:30 pm
+
== 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.
-
* Transformed J23100 A + S01414 A, J23100 B + S01414 B, J23100 C + S01640 B in DH5A cells.
+
== October 20 ==
-
** J23100 D + S01640 A was not transformed because there are only three AMP plates.
+
(Anam)
-
* MP o/n F1610 AB from the plate Seema gave us (part was from 2006 plate).
+
* miniprepped T9002(AB), E1A(AB)
-
** Incubating o/n in shaker.
+
* quantitated Q04400B: no bands seen
 +
* length checks of T9002, N4, N5: all pass
-
'''TO DO:'''
+
== October 19, 2007 ==
-
* MP F1610 and do PD length check for it.
+
-
* 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
+
Yusuf, Maria, Faareha, Talal
-
Start time: 6:30 pm
+
Start: 3pm - 12am
-
* Created stocks of parts to store in -80 freezer.
+
'''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
-
PROCEDURE:
+
* Digest
-
* Did MP o/n yesterday
+
** Q04400 B - primary digest with X/P wasn't successful. Only plasmid was seen. Second digest with E/S
-
* Added 500 uL of MP o/n solution in eppendorf tube
+
*** If digest of Q04400 has been ok ed by charles proceed to Quantitation and ligation and transformation
-
* Added 500 uL of Glycerol in eppendorf tube & mix well
+
** Enzyme check for SpeI - all enzymes working
-
* Filled 5 eppendorf tubes per part
+
-
== August 30, 2007 ==
+
'''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
-
=== Morning Session ===
+
== October 18th, 2007 ==  
-
'''Start time: 2:30 pm'''
+
Natalie
-
F/T: Yusuf
+
*Rechecked Q04400B - everything is OK (verified by Seema and Esther).
-
* Since the part '''F1610''' was not working from the iGem 2007 plate, we had to borrow this part from Seema whose '''F1610''' part was from iGem 2006 plate and presumably it worked for her. So I did transformation for the part '''F1610''' and left it for overnight in the incubator.
+
Esther, Neha, Rafsan
-
* Tomorrow, 31st August, 2007, I have to do Mini prep o/n for '''F1610'''
+
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).
-
* I have filled up all the Pipette Tips and will drop off at the Autoclave room to be sterilised and we later need to pick them up tomorrow morning.
+
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.
-
* Things To Do for the EVENING SESSION:
+
3. PD of N4 A,B and N5 A,B - used X/P
-
** Make stock cells for the parts that had successful length checks
+
4. Quantitation:
-
** Ligation for '''J23100''' with '''S01640''' and '''S01414'''
+
*I13033
 +
*N1A
 +
*R0082 #1
 +
*I13504A
-
* Things To Do TOMORROW BEFORE GROUP MEETING AT 12PM  AT MED-SCI CAFETERIA:  
+
Note: The gel used was yesterday's.
-
** DEPENDS ON HOW MUCH WE ARE DONE TODAY
+
Results:
-
=== Evening Session ===
+
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.
-
F/T: Anam
+
To do:  
-
P/T: Rafsan, Fareeha
+
-
* MP o/n for all parts except for F1610, J23100 (one colony each).
+
1. MP O/N of E1b, N1
-
* Performed quantitation for parts J23100 ABCD, S01414 AB, S01640 AB.
+
2. PD check: PD of N4 A,B and N5 A,B
-
** Got data for J23100 ABD, didnt get data for the rest because the dna was too dilute (since I accidently put in 50 uL of TE buffer) and so didnt show up on the gel (or shows extremely light.
+
3. Quantitate:
-
*** TO YUSUF: I will finish up quantitation and ligation before morning team comes in tomorrow.
+
*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).
-
== August 29, 2007 ==
+
== October 17th, 2007 ==
-
=== Morning Session ===
+
Yusuf (5:00pm - 10:00pm)
-
'''Start time: 11 am'''
+
'''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)
-
F/T: Anam
+
'''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)
-
P/T: Talal (1:30pm to 4:30pm)
+
== October 16, 2007 ==
-
* MP J37033 colonies A’’, B’’, C’’, D’’ & F1610 colonies A’’, B’’, C’’, D’’
+
Natalie (6:30 - 12:30)
-
* Froze pellets of J37033 colonies A’’’, B’’’, C’’’, D’’’ since doing MP on this right now would unnecessarily waste MP kit materials (Seema has expressed concern on the point that we are going through the Fermentas MP kits too fast). Will wait to fully MP this after PD length check results. If the J37033 colonies A’’, B’’, C’’, D’’ pass the length check, we don't need to MP this.
+
-
* PD digest of J23100 ABCD (used enzymes S/P for plasmid), S01414 AB (used enzymes X/P for insert), S01640 AB (used enzymes X/P for insert). Used following recipe for PD:
+
-
** 21 µL of plasmid, 3 µL of BSA, 3 µL of Buffer, 1.5 µL of enzyme1, 1.5 µL of enzyme2
+
-
** Used buffer 2 for all PD.
+
-
* Gel run for all performed.
+
-
* Turned out okay, but the total volume for the recipe used today was a bit too much for the small wells. Next time only use the amounts given on the PD recipe on protocol multiplied by 2.
+
-
* Extracted parts from gel, purified it, and stored it in the -20 freezer for ligation tomorrow.
+
-
**Note: Used 50 uL of TE buffer during gel purification instead of 30 uL. Will use 30 uL next time because it concentrates the DNA.
+
-
===Evening Session===
+
-
P/T: Maria and Daniel '''(5 pm)'''
+
From yesterday: E1A transformation unsuccessful; no colonies
-
* PD of J37033 colonies A’’, B’’, C’’, D’’ & F1610 colonies A’’, B’’, C’’, D’’ using enzymes X/S
+
Today:
-
* Ran gel for PD length check
+
* ligation of R0062 and E0433 (tube in freezer labelled "nat1")
-
 
+
* ligation of I0462 and (R0062 + E0240) (tube in freezer labelled "nat2")
-
====Results====
+
* Seema plated T9002 in DH5a
-
 
+
* stored half a clean gel in the fridge
-
F1610 (for all colonies): Band 1 -> 6-7 OR 7, Band 2 -> 14
+
* digested Q04400B and I13033 for length checking; stored gel in fridge for second-party verification
-
 
+
** O = match, X = non-match
-
* Saw plasmid, but not part even though we should have been able to see it. Saw a band of a very small size which was unusual and unexpected.
+
{| border=1
-
 
+
! Part Name !! Part !! Plasmid
-
J37033 (for all colonies): 8-9 OR 9
+
|-
-
 
+
| Q04400B || O || O
-
* Saw only plasmid, but not part even though we should have been able to see it.
+
|-
-
 
+
| I13033 || X? || O
-
====TO DO (regarding PD length check)====
+
|}
-
 
+
** may need to find part for I13033 using more finely grained ladder
-
* Check this unexpected part of small size is actually a subpart of F1610.
+
* made 3 extra AMP plates and stored the rest of the agar in a different beaker
-
* Start looking for subparts to build F1610 from.
+
* prepared the plasmids from Calgary for transformation (pPCB, pCph8)
-
* J37033 has two other working analogs which can substitute for it (S01414, S01640)
+
** more like attempted! Will find out if it worked tomorrow... thanks, Patrick!
-
 
+
* transformed parts into DH5a cells and plated:
-
== August 28, 2007 ==
+
** 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)
-
===Morning Session===
+
To do tomorrow:
-
'''Start time: 11:00 a.m. - End time: 2:30 p.m.'''
+
* 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
-
Members present: Yusuf, Talal
+
== Oct. 15th, 2007 ==
 +
yusuf, Irina
-
# Carried out mini-prep of F1610
+
* transformation of (J23100+S01640) + J06801 labelled as E1A
-
#* Four colonies labeled A’ , B’ , C’ and D’ were selected
+
** used 4μl of ligated material from iGEM 2007 box
-
#* Then, plasmid digestion and subsequent length check was carried out
+
** plate used AMP+Kan
-
#* '''Results:''' 1st band (bright) between 5 & 6 (right in the middle). This band makes sense for part + plasmid. 2nd band (faint) between  13 & 14 (closer to 13). Doesn’t make sense.
+
-
# Miniprep overnight carried out for J37033
+
-
#* Four colonies A’’, B’’, C’’, and D’’ were selected and incubated overnight in shaker.
+
-
===Evening Session===
+
* MP of N2 (A,B,C,D) and length check
-
'''Start time: 5pm'''
+
** 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
-
Members: Anam, Daniel, Maria
+
* LENGTH CHECK RESULTS:
-
 
+
{| border=1
-
* MP o/n for J37033 using newly transformed cells (this part is giving us trouble…maybe if we check a lot of different colonies at the same time, we might get lucky since it increases our chance of finding the sample from the right colony)
+
|+ N2 Results
-
* Four colonies A’’’, B’’’, C’’’, D’’’ incubated overnight in shaker
+
! Part Name !! Part !! Plasmid
-
* MP o/n of F1610 using different colonies from the same plate that had been transformed before by Yusuf and Charles
+
-
* Four colonies labeled A’’, B’’, C’’, D’’ incubated in shaker overnight.
+
-
* Made 1 X TAE Buffer since we were out.
+
-
 
+
-
== August 27, 2007 ==
+
-
 
+
-
===Morning Session===
+
-
'''Start Time: 11:00 pm – End Time: 3:00 pm'''
+
-
 
+
-
Members Present: Yusuf, Mimi, Talal
+
-
 
+
-
* Carried out plasmid digest (length checks) on I 13507 B and J 37033 B
+
-
* '''Length Check results:'''
+
-
** I 13507 B – ''Overall 3 bands were seen for this plasmid''
+
-
*** 6-7 (Closer to 7 than to 6)
+
-
*** 8-9 (Closer to 9 than to 8)
+
-
*** 11-12 (Closer to 11 than to 12)
+
-
*** The number of parts and parts and plasmid match up closely but the first band between 6-7 could be of the uncut plasmid
+
-
** J 37033 B – ''Overall 2 bands were seen''
+
-
*** 8-9 (Closer to 9 than to 8)
+
-
*** 10-11 (In the middle)
+
-
*** The bands formed doesn’t match up with the given numbers for its part and plasmid from the registry
+
-
* Did Mini-prep overnight for F 1610  with 4 colonies labeled A', B', C', D' and put it in the incubator
+
-
 
+
-
===Evening Session===
+
-
'''Start time: 5pm - End time: 9:30 pm'''
+
-
 
+
-
F/T: Anam
+
-
 
+
-
* Performed DH5(alpha) + J37033 transformation since none of the PD length checks for all four colonies was odd and the last transformation showed only 5 big, dense circles with very tiny dots around each one even though it was not left for more time than the other transformation. This odd growth might be due to a problem with the transformation. Possible fungus growth? (Seema has informed me that there is a chance that they could grow during the transformation)
+
-
 
+
-
 
+
-
{| border="1"
+
-
|+ Plasmid Length Check Results
+
-
|-
+
-
! &nbsp; !! I13507 !! F1610 !! S01640 !! S01414 !! J37033¹ !! J23100² !! S01003
+
|-
|-
-
! A
+
| A   ||   N/A  ||   x
-
| 1000-750 (saw 3 bands) || '''4000'''-3500, 500-'''250''' || 1000-'''750''' || 1000-'''750''' || '''5000'''-4000, 2000-'''1500''' || 3000 || '''1000'''-750 (saw 3 bands)
+
|-
|-
-
! B
+
| B   ||   N/A  ||   x
-
| 3500-'''2500''', 2500-'''2000''', '''1000'''-750 || '''3500'''-2500, 500-'''250''' || 1000-'''750''' || 1000-'''750''' (saw 3 bands) || 2000-'''1500''', 1500-1000 || 3000-'''2500''' || '''3000'''-2500 (faint), 2000 (brightest), 1500-'''1000''' (2nd brightest)
+
|-
|-
-
! C
+
| C   ||   N/A  ||   x
-
| 3000-2500³, 2500-'''2000''', 1000-750 (middle) || 3000-2500(middle), 2000-'''1500''' OR 2500-2000, 500-'''250''' || '''3000'''-2500, 2500-'''2000''', 1000-'''750''' || 2500-'''2000''', 1000-'''750''' || 2500-'''2000''' || 3500-3000 || 3000-'''2500''' (faint), 2000 (brightest), 1500-'''1000''' (2nd brightest)
+
|-
|-
-
! D
+
| D   ||   N/A  ||   x
-
| 2500-'''2000''', 1000-750 (middle) || 3000-2500 (middle), 500-'''250''' || '''3000'''-2500, 2500-'''2000''', 1000-'''750''' || 2500-'''2000''', 1000-'''750''' || 2500-'''2000''' || 3000-'''2500''' || 3000-'''2500''' (faint), 2000 (brightest), 1500-'''1000''' (2nd brightest)
+
|}
|}
-
Legend:
+
* digest and length check for Q04400:
-
* / - means "in between"
+
** Enzyme used Xba/Pst
-
* '''Boldfaced''' – means "closer to"
+
** Not a match
-
* ¹ For Part J37033 colonies C and D, only one band was seen.
+
-
* ² Cannot see part, but saw plasmid for C and D for which Yusuf cut the part out
+
-
* ³ One person this while the other didn’t
+
 +
* To do:
-
'''Note from Anam:''' For colonies A of all parts, S02640 colony B, S01414 colony B, and F1610 colony B, I only wrote down the location of the band that had run furthest in the gel (except for the ones that weren’t correct – I recorded all the band locations for troubleshooting later) because all the parts are significantly smaller than their corresponding plasmids (Mistake on my part..I will write all the band locations in the future). I just wrote down how many bands I saw if there were more than 2 there.
+
** 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 ==
-
'''CONCLUSION from length check'''
+
Esther
-
All parts passed except for all colonies of F1610 and J37033.
+
1. MP O/N of N2 A, B, C, D.  
-
== August 24, 2007 ==
+
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.
-
===Evening Session===
+
To be done by Adnan in the afternoon:
-
'''Time: 5 pm'''
+
-
F/T: Anam
+
1. Ligation of J06801A and (J23100+S01640)
 +
*Both have been quantified and are in the enzyme box.
-
P/T: Talal
+
2. PD and GE of
 +
*I13033
 +
*N1A
 +
*R0082 #1
 +
*I13504A
-
* Record the results of the gel Yusuf had made and run (included in table below)
+
To do (Monday):
-
* PD length check for J23100 colony C successful.
+
# Transformation of J06801A+(J23100+S01640)
-
* 1 band between 6 and 7 on gel
+
# MP of N2 A,B,C,D
 +
# PD check and then PD, GE of N2
 +
# ligations from #2 (check flow chart)
-
== August 23, 2007 ==
+
== Oct. 13th, 2007 ==
-
===Evening Session===
+
Anam
-
'''Start time: 6:30pm'''
+
-
F/T: 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).
-
P/T: Fareeha
+
*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.
-
* Done length check PD for all of A, green circle B, red circle B, black dot B
+
== Oct. 12th, 2007 ==
-
* Enzymes X and P used for all except for J23100 because the part is too small and so the plasmid that contained it was linearized using enzyme P.
+
-
* Buffer 2 was used.
+
-
====Results====
+
Yusuf, Faareha, Talal
-
* All were correct length except for samples from colonies A and B of F1610 and colony A of J37033.
+
-
* S01003 (colony A), S01414 (colony B), I13507 (colony A) had 3 bands.
+
-
* The 3rd band might have shown up because some of the plasmid was only cut in one location.
+
-
====To do list====
+
* Transformation of N2 (New) into (AMP + KAN) plate.
 +
** It's in incubator.
-
# Do a PD length check for the rest of the samples
+
* MP of Q04400 (A,B)and I13033.
-
# Discuss with Yusuf how much information needs to be recorded when observing the bands in a gel for PD length check
+
-
''Note:'' There is one gel left in the fridge. It was made today.
+
* Length check for Q04400 (A,B).
-
== August 22, 2007 ==
+
* Length check for R0082 (A,B)
 +
** Part length: 108 bp
 +
** Plasmid length: 2079 bp
-
===Evening Session===
+
* LENGTH CHECK RESULTS:
-
'''Start Time: 5:30 pm – End Time: 7:30 pm'''
+
{| 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
 +
|}
-
Members Present: Yusuf, Anam
+
* '''YEAHHH WE HAVE GOT QO4400 NOW WE CAN START OUR WORK''' 
-
* We did Mini Prep overnight and since I did 7 transformations, we decided to do 4 copies of each labeled A,B,C,D so that we have more flexibility in choosing our parts properly.
+
* Enzyme activity
-
* These are the following symbols we used to clarify all the tubes with the parts
+
** Recipe used:
-
** Black circle - I 13507 (AMP Res) (A – D)
+
*** Enzyme: 0.25 μL
-
** Red circle - F 1610 (AMP & KAN Res) (A – D)
+
*** Plasmid (J04500): 1 μL
-
** Green Dot - S 01640 (AMP Res) (A – D)
+
*** BSA: 0.5 μL
-
** Black dot - S 01414 (AMP Res) (A – D)
+
*** Buffer: 0.5 μL
-
** Red dot - J 37033 (AMP Res) (A – D)
+
*** Distilled water: 3.75 μL
-
** Black line - J 23100 which is a promoter (AMP Res) (A – D)
+
-
** Red line - S 01003 (AMP Res) (A – D)
+
-
* We also labeled our colonies from the Agar plates which were chosen for miniprep overnight as it was instructed by Seema to both Anam and Esther.
+
-
* The cells which had the promoter J 23100 were light pink colored; if Charles and Andy would like to shed some light upon that, that would be great.
+
-
== August 21, 2007 ==
+
**RESULTS IN LAB NOTEBOOK: RESULTS PRETTY GOOD FOR ALL OF THEM
-
===Morning Session===
+
TO DO:
-
'''Start Time: 11:00 am'''
+
* LIGATE R0082 WITH E0240 A
 +
** TRANSFORM THE LIGATED CONSTRUCT
-
* Charles showed me how to use the registry properly so if anyone needs to know  how to use the registry properly just let me know.
+
* MP O/N FOR N2
-
** We searched for super parts which is more like a couple of parts ligated together so that it makes our job a lot easier
+
-
** We chose 7 parts to be transformed from the neural network MODIFIED model
+
-
* Part timer Muhammad Talal Latif showed up and I instructed him on how to do transformation properly
+
* DIGEST, GEL EXTRACT, PURIFICATION OF Q04400
-
* So I used 20 µL of TE buffer to extract:
+
-
** J 37033: 8F, Plate:3 (Amp Res)
+
-
** S 01640: 3L, Plate:3 (Amp Res)
+
-
* Both of them were from the iGEM 2007 Kit Plates
+
-
* We stored the plasmids in the –20°C freezer labeled in red DNA remaining
+
-
* Added 4 µL  of J37033 and S01640 to separate competent cell (DH5αZ1) eppendorf tubes from the –80°C freezer
+
-
* So now we are following the Transformation steps from the guidelines posted
+
-
* We used two AMP plates made from before
+
-
* We also made 8 AMP plates where 4 is going to be used today for the other four parts which we also transformed:
+
* RECHECK LENGTH CHECK FOR I13033 BECAUSE THE PLASMID SHOWS UP BUT THE PART DOESNT
-
** I 13507: (Used form 2005 stock) (AMP Res)
+
* WE HAVE A LOT OF AGAR GELS IN THE FREEZER USE THEM
-
** S 01003: 20O, Plate:1 (AMP Res)
+
-
** J 23100: 21E, Plate: 3 (AMP Res)
+
-
** S 01414: 22O, Plate: 1 (AMP Res)
+
-
* We also made 3 AMP and KAN plates where 1 is going to be used:
+
== Oct. 11th, 2007 ==
-
** F 1610: 1B, Plate: 2, (AMP and KAN Resistant)
+
Esther, Neha
-
* CONFUSION: While making the AMP and KAN plates we used AMP of 100mg/ml whereas KAN was 30mg/ml. So even though KAN was of different concentration we used the same quantity.
+
1. MP O/N of
-
** CHARLES AND ANDY GIVE US SOME INSIGHT ON TO THIS TOPIC
+
*Q04400
-
* So we left all 7 plates for overnight incubation and hopefully we see some results tomorrow
+
**6 tubes of colony A
-
* Everybody please welcome SeungMe (part time) who came in and helped me finish the work today and of course Charles
+
**6 tubes of colony B
 +
*I13033
 +
**6 tubes of colony A
 +
*Each tube contained 1mL of LB
-
== August 13, 2007 ==
+
2. PD of E0240A and Q04400B (old MP), along with enzyme checks (see lab notebook)
-
MP and sample PD and gel run.  
+
3. Ligation of N1A and S01003. (N1A + S01003 = N2)
 +
4. MP of J06801 A&B
-
10 MP total:
+
To do:  
-
* 5 MP of R0040+E0240 (Black): A, B, C, D, E
+
-
* 5 MP of R0040+E0240 (Red): A, B, C, D, E.
+
 +
1. Recheck the relative efficiency of all enzymes in the box.
-
Sample PD was done and gel run.  
+
2. Sample PD for R0082.
 +
*if good, takes steps to ligate with E0240A
 +
*if not good, troubleshoot - remake the MP with different colonies, etc.
-
Enzymes used: X/P
+
3. MP of Q04400 (A and B) and I13033.
-
Three bands for all:
+
4. Transformation of N2
-
~3000, ~2000-2500, ~1000.
+
== Oct. 10th, 2007 ==
 +
Yusuf
-
Presumably they are undigested plasmid, plasmid alone, and promoter+reporter unit.
+
* Transformation of Q04400 in DH5a from DNA of 2006
 +
** Its in the incubator and needs to be MP o/n tomorrow
-
== August 12, 2007 ==
+
* Quantitation of N1 and S01003 using previously digested parts
-
MP O/N
+
** '''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'''
-
5 colonies for both petri dishes of R0040+E0240.
+
* 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
-
Colonies were labelled A, B, C, D, E
+
*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
 +
|}
-
Aug. 11/07 ligation dish was labelled in BLACK.
+
* Mp o/n of J06801
 +
* I13033: plate couldnt be found
-
Aug 10/07 (uncertain) ligation dish was labelled in RED.
+
* 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
-
Tomorrow: MP
+
== Oct. 9, 2007 ==
-
''Note:'' DO NOT use the AMP in the iGEM box, as it was left out overnight at room temperature. It may have degraded (meaning, it may be unusable).
+
* 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):
-
In addition:
+
{| border=1
-
* Eppendorf tubes should be put into Eppendorf tube holding tray, not the test tube holding tray.
+
! Part Name !! Part !! Plasmid
-
* Also, make certain you put everything you took out of the fridge back in. Thanks.
+
|-
 +
| N2 A || X || O
 +
|-
 +
| N2 B || X || O
 +
|-
 +
| N2 C || X || O
 +
|-
 +
| N2 D || X || O
 +
|-
 +
|}
-
== August 11, 2007 ==
+
* 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)
-
Ligation + Transformation:  
+
'''To Do:'''
-
* Ligation of R0040 and E0240 done with standard procedures.
+
-
* Transformation of both today’s ligation and yesterday’s uncertain ligation (marked in RED) for two petri dishes total.
+
-
== August 10, 2007 ==
+
* 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
-
PD, GE, and ligation
+
== Oct. 8, 2007 ==
-
PD and GE done with standard procedures.
+
Yusuf
-
Ligation could have been unsuccessful due to incubation at 37°C instead of room temperature.
+
* 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"
-
'''Tomorrow:'''
+
TO DO:
-
* Religate
+
-
* Transformation
+
-
== August 9, 2007 ==
+
* Make
 +
** 5 AMP plates
 +
** 5 KAN Plates
 +
** 3 AMP+KAN Plates
-
MP and sample PD run on R0040.
+
* Mini prep of:(in incubator)
 +
** E1B# 1 -> A&B
 +
** E1B# 2 -> A&B
-
'''Tomorrow:'''
+
* Length Check for:
-
* PD + Gel extraction
+
** E1B# 1 -> A&B
-
* Quantitation
+
** E1B# 2 -> A&B
-
* Ligation
+
** N2 -> A,B,C,D
 +
** R0062 -> C,D,E,F
-
== August 8, 2007 ==
+
* Plasmid Digest and gel extract of the above constructs
-
PD of R0040. Mistake made post-gel extraction where all the purified DNA was mixed with loading dye. This mixture is in the –20 freezer at the moment.  
+
== Oct. 7, 2007 ==
-
MP O/N was prepped with R0040 A from petri dish.
+
Esther, Conrad, Adnan
-
'''Tomorrow:'''
+
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)
-
Starting at 12 PM:
+
To Do:  
-
# MP (1-2 hrs), make gel while MP.
+
*MP O/N of E1b
-
# make more agar (while MP)
+
*MP of N2 (A,B,C,D) and R0062 (C,D,E,F)
-
# PD test run (1 hr at most)
+
* Digest, GE, purify, quantitate:
-
# PD + gel purification/extraction (2-3 hrs)
+
** I13033 A, S/P
-
# quantitation + ligation (2 hrs)
+
** J06801 A, E/X
 +
** Q04400 C, X/P (assuming R0011 was cut with S/P)
 +
* PD length check I13504 AB
-
If you’re coming in tomorrow, I recommend you pack lunch.
+
Note: We are out of EtBr. Check with Seema.
-
== August 7, 2007 ==
+
== Oct. 6, 2007 ==
-
Made stock cells of R0040 and R0011+E0240.
+
Anam, Tara, Jovan
-
* L1/A: 4 tubes
+
-
* L1/B: 4 tubes
+
-
* L2/A: 4 tubes
+
-
* L2/B: 4 tubes
+
-
* R0040: 4 tubes
+
-
They are in the –80°C freezer.
+
*Finished gel purification of J06801, J31003, B0034, E0433, I13033
 +
*MP for Q04400CD, R0062AB
 +
*PD length check for Q04400 CD, R0062AB
-
== August 6, 2007 ==
+
{| 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)
-
MP + Test PD
 
-
===MP===
+
'''TO DO:'''
-
* 5 tubes:
+
* Transform E1b
-
** R0011+E0240 L1 A,B
+
* MP I13033A, and J06801A (and make stock if there isn't any in the -80 freezer of this)
-
** R0011 +E0240 L2 A,B
+
** Don't do PD length check on these because it has already been done before and has passed it.
-
** R0040
+
* 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)
-
MP was done as usual, except for R0040, 0.5 mL was used to make stock (50/50 mix with glycerol).
+
== Oct. 5, 2007 ==
-
===PD test run===
+
Yusuf, Maria, Talal, Faareha
-
Ligated material:  
+
'''SUMMARY:'''
-
* Standard proportions used.
+
-
* Enzymes: X/P
+
-
* Total volume: 10mL
+
-
R0040:
+
* Miniprep: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
-
* Standard proportions.
+
* Length Check:
-
* Enzyme: P
+
{| border=1
-
* Total volume: 10mL
+
! Part Name !! Part !! Plasmid
-
* Results:
+
|-
 +
| 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
 +
|}
-
====Expected====
+
* 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
-
R0040:
+
'''TO DO:'''
-
* Part: 54 bp
+
-
* Plasmid: 2079
+
-
* Part and Plasmid: 2133
+
-
R0011:
+
* B0015 (2005) didn't transform, try again
-
* Part: 55bp
+
* Complete gel extract procedure
-
* Plasmid: 2079
+
* Ligate:
-
* Part and Plasmid: 2134
+
** B0034 A + J31003 A
 +
** I13033 A + E0433 A
 +
** (J23100 + S01640) D + J06801 A
-
E0240
+
== Oct. 4, 2007 ==
-
* Part: 879bp
+
-
* Plasmid: 2079
+
-
* Part and Plasmid: 2955
+
-
R0011+E0240
+
Esther, Neha, Rafsan
-
* Uncut: plasmid+promoter+gene: 3013
+
-
R0040:  
+
'''SUMMARY:'''
-
* One band b/t 2500/2000.
+
-
R0011+E0240:  
+
* Found alternate to I1303: ligate terminator (B0015) and Lux pR promoter (R0062) separately
-
* Optimal: Two bands, 2000bp and 1000bp
+
* 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
-
Possible:  
+
'''TO DO:'''
-
* One cut, gene+promote+plasmid
+
-
* Two cuts, gene+promoter, plasmid
+
-
+
-
====Actual Results====
+
-
* R0040 adhered to expected results.
+
-
* Ligated material had anomalies:
+
-
** L1/A: 1500 bp band = ?
+
-
** L1/B, L2/A,B: 750 bp band = ? GFP gene alone?
+
-
MP O/N done for all five.
+
* 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
-
== August 5, 2007 ==
+
== Oct. 3, 2007 ==
-
MP O/N:
+
Yusuf
-
* Five tubes
+
-
** 1 x R0040/DH5a (A)
+
-
** 2 x R0011+E0240 L1/(A), (B)
+
-
** 2 x R0011+E0240 L2/(A), (B)
+
-
Standard procedures were used. The AMP used is now in the iGEM box.
+
'''SUMMARY:'''
-
Future plans:  
+
* Transformed: J06801 (2005), Q04400 (2005), B0034 (2007), J31005 (2007)
-
# MP for all items.
+
* Minipreped I130033 AB, E0433 AB, S03140 AB, J04500 AB, J31003 AB, N1 AB
-
# stock tube for E0040.
+
* Made o/n of DH5a and DH5a-z1 (from July 16 plate - ask Seema if this is ok)
-
# If  time allows:  
+
* Length Check (O = match, X = does not match):
-
## Standard PD/gel/quantitation/extraction for R0040.
+
{| border=1
-
## Test PD and gel run for ligated material
+
! Part Name !! Part !! Plasmid
-
### Use S/P to cut out the insert; if the entire process was successful, the gel should show bands for both the R0011 and the E0240.
+
|-
 +
| 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
 +
|}
-
Making Stock:  
+
'''TO DO:'''
-
Add same volume of glycerol as in left over MP tube. For this stock, it will be 0.5mL of MP and 0.5mL of glycerol (the glycerol is to protect the cell membranes whilst in the fridge).
+
* 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
-
== August 4, 2007 ==
+
== Oct. 2, 2007 ==
-
===Session===
+
Natalie
-
Did 3 transformations:  
+
'''SUMMARY:'''
-
# E0240+R0011 into DH5aZ1, into AMP plates.
+
-
# E0240+R0011 into DH5aZ1, into AMP plates.
+
-
# R0040 into DH5a, into AMP plates.
+
-
E0240+R0011 were taken from Aug. 3, 2007 ligated stock.
+
* 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
-
R0040 directly from iGEM 2007 plate 1, well 70.
+
'''TO DO:'''
-
 
+
-
For transformations 1 and 2, standard procedures were used with small time variations.
+
-
 
+
-
For transformations 3:  
+
-
# Extraction from the plate was done using 15mL of elution buffer, not deionized water.
+
-
# Commercial DH5α was used from Seema’s store to increase likelihood of success.
+
-
# Otherwise, standard procedures were used.
+
-
 
+
-
Further notes:
+
-
# We require more AMP plates. Whoever is in on Tuesday must make the plates. Remember you need to make the agar right before the autoclave cycle. Time it accordingly.
+
-
#  Should we be starting a cloning process with DH5αZ1 if DH5α is successful? Consider uses.
+
-
 
+
-
===Future Plans===
+
-
* Sunday: MP o/n prep.
+
-
* Monday: MP, PD test run + gel test run
+
-
* Tuesday: Actual PD + gel extraction + quantitate
+
-
* Wednesday: ligation + transformation
+
-
 
+
-
== August 3, 2007 ==
+
-
 
+
-
We did three things today:
+
-
# Plasmid Digest
+
-
# Gel Run + Quantitation + Gel Extraction 
+
-
# Ligation
+
-
 
+
-
Plasmid and insert (two identical copies each) were run through #1 and #2, and ligated together during #3 (final count: two tubes of ligated material).
+
-
* Plasmid: iGEM 2006 R0011, colony ?
+
* Miniprep all above parts and length check
-
* Insert: iGEM 2006 E0240, colony ? X2
+
* 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)
-
As the materials used were from a successful sample PD from the day before, the entire thing was put through step #2 without a sample PD/Gel run.  
+
== Oct. 1, 2007 ==
-
Quantitation:  
+
'''Time: 3:45 PM - 8:30 PM'''
-
* R0011: 9 ng/mL
+
-
* E0240: 2 ng/mL
+
-
Ligation:
+
Yusuf, Irina
-
* R0011 (10 ng): 1mL
+
-
* E0240 (12 ng): 6mL
+
-
* 10x buffer: 1 mL
+
-
* ligase: 0.5 mL
+
-
* ddH₂O: 1.5 mL
+
-
Plasmid, insert, and water were added first into one Eppendorf tube, then buffer and ligase. Standard procedures were followed after this point.
+
Summary:
-
There were two copies of ligated material.
+
* made 5 KAN plates
 +
* made 2 AMP+KAN plates
 +
* made 300 mL LB
-
<small>
+
Tranformation of following parts:
-
Back to the [[Toronto|Toronto team]] main page
+
* I13033 -> KAN
-
</small>
+
* E0433 -> KAN
 +
* S03140 -> AMP
 +
* J04500 -> KAN
 +
* J31003 -> AMP
 +
* (J23100C + S01640B)+ B0015 -> KAN
-
== Headline text ==
+
Used 4μL of DNA for all transformations
-
== Headline text ==
+
* 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 !!!
-
== Headline text ==
+
TO DO:
-
== Headline text ==
+
* MP o/n for the above parts

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
Retrieved from "http://2007.igem.org/wiki/index.php/Toronto/Lab_Notebook"