Team:Monash Australia/Notebook
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==12/8/10== | ==12/8/10== | ||
+ | <b>Test of PCR product of SAM Synthetase + Cont of seed stock</b> | ||
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+ | === PCR === | ||
+ | |||
+ | * Made up 1% agarose Gel. | ||
+ | * Used Easy vision DNA marker to both ladder and PCR product. | ||
+ | * DNA ladder used = Gene ruler DNA ladder by Fermentas. | ||
+ | * Ran 5 μL of PCR product and DNA marker for 30 minutes at 80 V. | ||
+ | |||
+ | * Attach Gel photo here! | ||
+ | |||
+ | |||
+ | === Seed stock === | ||
+ | |||
+ | * Made up stock soln. of glycerol, 30% @ 20 mL. | ||
+ | * Added steptomycin to SOB stock. | ||
+ | * Created 5 tubes of seed stock. | ||
+ | * Stocks are shaken at 23 ℃ overnight. | ||
+ | * Places 5 seed stocks in -80 ℃ freezer. | ||
==Date== | ==Date== |
Revision as of 11:13, 22 August 2010
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Contents |
Lab Notebook
09/08/10
Created 1L SOB media using:
- 5g yeast extract
- 0.5g NaCl
- 0.19g KCl
- 20g Tryptone
of the 1L of SOB media, 2x 250ml was autoclaved; one was used to make agar medium the other was used for medium. The other 500ml was left for storage, however was subsequently destroyed due to contamination.
3.75g of agar was added to 250ml of SOB media to produce agar media
Created a batch CCMB80 Buffer as followed:
- 2.5ml KoAc
- 2.95g CaCl2.2H2O
- 1g MnCl2.4H2O
- 0.5g MgCl2.6H2O
- 25ml Glycerol
- HCl to bring pH ~ 6.4
- Poured 4x SOB Agar plates.
- Poured 3x SOB Agar plates with Streptomycin.
- Streaked seed stocks of Top 10 cells on SOB Agar and SOB Agar w/ Strep.
- Grew at room temp for 48 hours.
10/8/10
- Amplified SAM Synthetase from E. coli genomic DNA using designed primers that incorporated biobrick prefix and suffix for RFC10.
PCR Protocol:
10x Buffer for KOD DNA Polymerase | 5 μL |
25mM MgCl2 | 3 μL |
dNTP's (2 nM each) | 5 μL |
PCR grade water | 28.1 μL |
Forward primer (5 pmol/μL) | 4 μL |
Reverse primer (5 pmol/μL) | 4 μL |
Purified E. coli genomic DNA | 0.5 μL |
KOD DNA Polymerase | 0.4 μL |
Total | 50 μL |
PCR Cycle:
Denaturation | 15 seconds @ 95 ℃ |
Annealing | 30 seconds @ 68 ℃ |
Elongation | 30 seconds @ 72 ℃ |
Repeat | 30 cycles |
Prepared a 100 μM stock solution of both primers to get 5 pmol/μL
11/8/10
Preparation of seed stock.
- Poured 27 mL of SOB media in sterile falcon tube to avoid contaminating the master batch.
- 5 mL of SOB media into 3 sterile falcon tubes.
- 5 μL of streptomycin in each tube.
- Innoculated each tube with single colonies from the previously cultured SOB Agar plate with strep.
- Mixed tubes.
- Places on shaker at 23 ℃/room temp.
12/8/10
Test of PCR product of SAM Synthetase + Cont of seed stock
PCR
- Made up 1% agarose Gel.
- Used Easy vision DNA marker to both ladder and PCR product.
- DNA ladder used = Gene ruler DNA ladder by Fermentas.
- Ran 5 μL of PCR product and DNA marker for 30 minutes at 80 V.
- Attach Gel photo here!
Seed stock
- Made up stock soln. of glycerol, 30% @ 20 mL.
- Added steptomycin to SOB stock.
- Created 5 tubes of seed stock.
- Stocks are shaken at 23 ℃ overnight.
- Places 5 seed stocks in -80 ℃ freezer.