Toronto/Lab Notebook

From 2007.igem.org

Latest revision as of 04:01, 27 October 2007

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

• • 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:

• 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:

• 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:

• 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):

• 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

• 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:

• 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):

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