Melbourne/Lab Notebook
From 2007.igem.org
Contents
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Week 1
- 25 June 2007: Prepared LB agar plates Amp & Kana.
- 25 June 2007: Resuspended the following from Registry plates & Transformed (shorter protocol) into competent DH5alpha cells.
- 26 June 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
- 26 June 2007: Streaked the following cells:
- pJS010 (from solid agar, Amp)
- Fusion protein (from glycerol stock, Amp?)
- BBa_I15010 (from solid agar, Kan)
- 27 June 2007: Transformed into Joe's competent DH5alpha cells.
Liquid culture
- Prepared 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
- Aliquoted 5mL Amp LB into 6 50mL falcon tubes
- To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
- To the Kan LB a single colony from the transformation plate of BBa_I15010 was introduced.
- Cells incubated at 37degrees with shaking overnight.
28 June 2007
Miniprep
- Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
- Stored in -20 freezer
Digest
Liquid culture
- Cultured the following cells from transformed plates:
29 June 2007
Miniprep
- Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
- Stored in -20 freezer
30 June 2007
Week 2
- 2 July 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
- Q04510
- E0241
- E0040
- B0014
- J61035
- 3 July 2007: Re Transformed into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.
- 4 July 2007:
Ampicillin Plates
- Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June.
- Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.
Tranformation
Transformed the following and grew on new ampicillin plates
- P1 5H
- P4 8J
- P2 15L
- Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)
Liquid Culture
- Cultured 2 colonies from each of the following transformed plates and labelled as follows
5 July 2007
Miniprep
- 1mL culture from the 4th of Julyput aside for glycerol stocks under sterile conditions. Labelled with todays date 5/7
- miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water. Labelled with todays date 5/7.
- the following liquid cultures were not miniprepped due to failure (no growth)
Digest
Performed the following digests on DNA from the above miniprep
EcoRI/PstI with buffer 3
EcoRI/HaeII in buffer 2
XbaI/SpeI in buffer 2
Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20
Transformation
- Transformed P1 11H from resuspended DNA.
Liquid Culture
6 July 2007
Digest Gel
- Prepared 20 lane 100mL agarose gel with 0.5xTBE buffer.
- Loaded 20uL of digest samples in the following lane order
- Ran for 1.5hours at 95V
Miniprep
- Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7
- Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.
- P1 5H 1
- P1 5H 2
- P4 8J 1
- P4 8J 2
- P2 15L 1
- P2 15L 2
Digest
- Digested 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.
- Incubated for 2hours 25min at 37degrees
- Added 5uL 6x loading dye and stored at -20
Glycerol Stocks
The following glycerol stocks were made:
- Put aside from cultures 5/7 (labelled with this date)
- Put aside from cultures 6/7 (labelled with this date)
Stored at -80
Liquid Culture
Cultured the following in 5mL LB
Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7
7 July 2007
- Made 10x TAE buffer
Digest Gel
- Prepared 8 lane 60mL agarose gel with 1xTAE buffer.
- Loaded 20uL of digest samples from 6/7 in the following lane order.
Glycerol Stocks
The following glycerol stocks were made and dated 7/7:
Stored at -80
Miniprep
- Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.
Digest
- Digested 5uL of each of the above miniprep DNA
- Incubated for 3hours at 37degrees
- Added 5uL 6x loading dye and stored at -20
8 July 2007
Transformation
Resuspended and transformed the following
- P1 15P(BBa_R0082, Omp R+, Amp)
- P1 17H(BBa_R0083, truncated BBa_R0082 Omp R+, Amp)
- P1 11A(BBa_E0430, EYFP(RBS+,LVA-,term) Amp)
- P1 16E(BBa_E0430; RBS,GFP,term; Amp)
The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.
- P2 13K (BBa_Q04510, c1 inverter, Kan)
Week 3
9 July 2007
- Digested I15010(E/P),P1-11H(E/H),P2-15L 1.5hrs run
- liquid culture P1-11H,I15010,P1-15P,P1-16E,P1-17H,P2-13K
10 July 2007
- Miniprep
- Digest P1-16E,P1-11A,P2-13K,I15010,P1-15P,P1-11H,P1-17H 1.0hrs run
- liquid culture I15010, P2
11 July 2007
- Digest for ligation P1-15P(1)10/7,P1-11H 10/7,P1-17H(1)10/7,P1-16E(2)10/7,P1-11A(1)10/7
- loaded order X/P P1-11A,X/P P1-16E,S/P P1-15P,S/P P1-11H
- Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.
- Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA
- Excise bands of interest and purify invitrogen
- liquid culture P1-11A,P1-15P 10ml
- Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.
- Glycerol stocks P1-11A,P1-16E,P1-11H,P1-15P,P1-17H
12 July 2007
- Ran Gel P1-11A,P1-16E,P1-15P,P1-11H
- miniprepped P1-11A,P1-15P
- Digest
- Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)
- Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1-11A),2uL H20)
- Liquid culture transformants 11/7
13 July 2007
- miniprep cultures from transformants 11/7
- Digestion P1-15P and P1-11A from 12/7/07 37degC 3hours 15 minutes stopped 5uL of 6X loading dye.
- Excise bands 800bp from P1-11A, 2Kbp from P1-15P and purified.
- Glycerol stocks of P3-20I,P2-21B,P2-23N
- Transform DH5a with ligation product
14 July 2007
- Transform using 10uL of ligation reaction
Week 4
16 July 2007
17 July 2007
18 July 2007
- Purchased XbaI,EcoRI,PstI
- Miniprepped cultures 1,3,6 from 16/7
- Digestion with E/P
- Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?
