Melbourne/Lab Notebook Weeks 1-4
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**[[Melbourne/BBa_E0040|'''P1 5H 2''']] | **[[Melbourne/BBa_E0040|'''P1 5H 2''']] | ||
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']] | **suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']] | ||
| + | *[[Melbourne/4 July 07 Digest|4/7/07 Confirmation of Miniprep Plasmids by digestion]] | ||
<font size=3><b>5 July 2007 | <font size=3><b>5 July 2007 | ||
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*[[Melbourne/Transformation Protocol|Transformed]] | *[[Melbourne/Transformation Protocol|Transformed]] | ||
| - | *#[[Melbourne/ | + | *#[[Melbourne/Lab Notebook gv 1|pN26]] |
| - | *#[[Melbourne/ | + | *#[[Melbourne/Lab Notebook gv 1|pN29]] |
with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways. | with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways. | ||
Latest revision as of 03:43, 25 October 2007
<Return to Lab notebook> <team home page>
Contents |
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
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
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.
Transformation
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
- 4/7/07 Confirmation of Miniprep Plasmids by digestion
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
EcoR1/Pst1 with buffer 3
EcoR1/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 (E/P) 1.5hrs run
- liquid culture P1 11H, I15010, P1 15P, P1 16E, P1 17H, P2 13K
Ran for 1.5 hours
10 July 2007
- Miniprep
- Digested P1 16E, P1 11A, P2 13K, I15010, P1 15P, P1 11H, P1 17H
- liquid culture I15010, P2 13K
Ran at 95V for 1 hour
11 July 2007
- Digested 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:
- Excise bands of interest and purify using invitrogen kit
- liquid culture P1 11A, P1 15P 10ml
- Transformed 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
(samples from gel purified DNA of 11/7)
-Did not work: too much DNA.
- 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
- Minipreped cultures from transformants 11/7
- Digested
from 12/7/07 37degC 3hours 15 minutes stopped by 5uL of 6X Loading dye.
- Ran on Gel for 1 hour
- 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 from 12/7.
14 July 2007
-results:
6 colonies on control plate
5 colonies on ligation plate
6 colonies on transformation of digested DNA from Thursday
Week 4
16 July 2007
- Colony PCR of all (5) colonies from ligation plate (14/7) and one from control plate.
- Digested same colonies at the same time to confirm that colony PCR was working.
-results: No colonies were apparent.
17 July 2007
- Set up repeat of Ligation from 12/7.
18 July 2007
- Purchased Restriction enzymes XbaI, EcoRI, PstI
- Miniprepped cultures 1,3,6 from 16/7
- Digested with E/P in triplicate.
- Ran Gel:
with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways.
-observe B+P plates clean. Small colonies on NL29 and NL26 plates as well as C.
- Picked three colonies from each plate and cultured.
20 July 2007
- Transformation of ligations from 17/7 together with a control of undigested P1 15P and competent cells only.
21 July 2007
- Transformation results from 20/7: 0 colonies on competent cells only control, many on undigested vector, and 3 on ligation plate.
