Toronto/Lab Notebook

From 2007.igem.org

Revision as of 21:01, 10 October 2007

PartsCatalogue

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
• 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/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:

• 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):

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