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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 |
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.
Preparation of competent cells
- Inoculated 250 mL SOB media with 1mL of seed stock and grown at room temp on a shaker.
- Started this at 6:00 pm 12/8/10.
13/8/10
Purification of PCR product and preparation of competent cells.
Purification of PCR product
- Used Wizard SV Gel and PCR clean up system to purify our suspected gene product.
- Followed the protocol that came with the kit.
- Places purified DNA into -80 ℃ freezer.
Preparation of competent cells
- Ben came into the lab at 10:00 am on 13.8.10 and observation of innoculated SOB medium looked well over an OD of 0.3
- He suspects that the shaker heated up overnight and sped up the growth.
- Remade 500 mL SOB media, 250 mL used in regrowth, 250 mL placed in storage. Both were autoclaved.
- Regrew cells in fresh medium.
- After an OD of 0.346.
- Centrifuged culture at 3,000 g's, 4 ℃ for 10 minutes.
- Resuspended cells in 80 mL filtered CCMB80 buffer.
- Incubated on ice for 20 minutes.
- Centrifuged at 4 ℃ and resuspended in 10 mL CCMB80 buffer.
- Incubated on ice for 20 minutes.
- Aliquot 100 μL of cell culture into sterile eppendorf tubes.
- Make note - You NEED a lot of sterile tubes, the protocol fails to mention this.
- Placed 100 or so ependorf tubes into -80 ℃ freezer.
16/8/10
Created more SOB and agar mixture and plated with ampicilin
- Using the protocol as stated on 9/8/10, created 500 mL of SOB agar.
- Autoclaved in 2x 250 mL flasks.
- Poured 4 agar plates.
- 60 mL SOB agar to 60 μL AMP.
20/8/10
Testing of comp. cells.
- Defrosted 400 μL of previously prepared comp cells.
- Rehydrated DNA from distribution kit with 10 μL of miliQ H2O.
- Transformed 50 μL of comp cells with:
- 1 μL PUC-19.
- 1 μL B0034 - RBS.
- 1 μL R0011 - LacI-Pl.
- 1 μL R0010 - LacI Native.
- 1 μL J13002 - p(tet) + RBS.
- 1 μL I13521 - p(tet) + mRFP.
- Hold on ice for 30 minutes.
- Heat shock at 42 ℃ for 60 seconds.
- Added 250 μL of SOC.
- Incubated at 37 ℃ for 1 hour in a shaking incubator.
- Plated 20 μL of each transformation plus a no DNA control on Amp plates.
- Incubated at 37 ℃ for 24 hours.
