Toronto/Lab Notebook
From 2007.igem.org
(Difference between revisions)
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** I13504 (A,B) | ** I13504 (A,B) | ||
*** For all these length checks enzyme combination Xba/Pst was used | *** 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 x | ||
+ | R0082 B x x | ||
+ | |||
+ | Made competent cells | ||
* Made: | * Made: |
Revision as of 00:09, 11 October 2007
Contents |
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
- 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 x R0082 B x x
Made competent cells
- 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
- 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. 9, 2007
- Digest, GE, purify, quantitate:
- I13033 A, S/P - Bands at: ~3500, ~3000 (cut out), 2000-1500
- [I13033] = 0 ng/uL
- J06801 A, E/X - Bands at: ~6000 (cut out)
- [J06801] = 0 ng/uL
- Q04400 C, X/P - Bands at: none
- I13033 A, S/P - Bands at: ~3500, ~3000 (cut out), 2000-1500
- 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.
- Label may have been switched between the two parts. Run a sample PD to check before proceeding.
- 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/uL
- [B0034] = 9.4 ng/uL
- [E0433] = 13.5 ng/uL
- 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/uL, S01003 (2007) = 4.1 ng/uL
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 4uL 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