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Team:Monash Australia/Notebook
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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 |
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- Prepared a 100 μM stock solution of both primers to get 5 pmol/μL
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