Bootcamp Protocols
Making LB for plates
To make 1 Liter of LB:
1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl.
2. Add dry ingredients first.
3. Use a 2L flask.
- Add the following
- 10 g Tryptone
- 5 g Yeast Extract
- 5 g NaCl
- 1.5 g Agar (NOT agarose!)
4. Then add 1 L of MilliQ water.
5. Autoclave by total volume.
6. Pour 25 mL on each plate (just enough to cover the bottom).
Making Liquid Media
Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl
Add dry ingredients first, then add MilliQ water
No Agar!
Autoclave by total volume
Making Glycerol Stock
Use flasks and bottles before graduated cylinders (they take forever to mix!)
Need a stir plate and a large stir bar
400 mL of 10% glycerol: 40 mL glycerol, 360 mL of MilliQ water
400 mL of 20% glycerol: 80 mL glycerol, 320 mL of MilliQ water
Don’t autoclave!
Making ddH2O
Use the small bottles. Autoclave water by total volume.
Autoclaving
Robert is the source of official help on all things about the Autoclave.
Look at the Betastar for settings. Go by materials and total volume.
Use the magical tape that changes color.
PCR Troubleshooting
Ran a diagnostic test to optimize buffer and temperature for the PCR. After doing the PCR (it’s nice to do this in linked PCR tubes) we run on a gel to test which worked
Buffers tested: GC + DMSO, HF + DMSO, GC, buffer G supermix + DMSO (3 tubes of each)
Made a master mix of primers, phusion, template DNA, and dNTPs. This was then used this for all the tubes of GC+DMSO, HF+DMSO, and GC.
Master Mix: 10 uL dNTP’s, 2 uL forward primers, 2 uL reverse primers, 2 uL template DNA, 1 uL phusion
GC + DMSO: 18 uL H2O, 6 uL GC buffer, 5.1 uL of master mix, 0.9 uL DMSO, now divide into 3 tubes (10 uL into each PCR tube)
HF + DMSO: 18 uL H2O, 6 uL HF buffer, 5.1 uL of master mix, 0.9 uL DMSO, now divide into 3 tubes (10 uL into each PCR tube)
GC: 18.9 uL H2O, 6 uL GC buffer, 5.1 uL master mix, now divide into 3 tubes (10 uL into each PCR tube)
Supermix: 15 uL buffer G, 12 uL H2O, 0.9 uL DMSO, 0.6 uL forward primer, 0.6 uL reverse primer, 0.6 uL template DNA, 0.3 uL phusion
PCR program:
1. 98 degrees Celsius for 3 minutes
2. 98 degrees C for 15 seconds
3. Temperature gradient 48-55-63 degrees for 30 seconds
4. 72 degrees C for 1 minute
5. Go to step 2, repeat 30 times
6. 72 degrees C for 5 minutes
Gel results: Only buffer G +DMSO works and it works at all temperatures.
Optomized PCR
Worked using buffer G +DMSO supermix at 63o C.
Supermix (for three reactions. 10uL/rxn): 15 uL buffer G, 12 uL H2O, 0.9 uL DMSO, 0.6 uL forward primer, 0.6 uL reverse primer, 0.6 uL template DNA, 0.3 uL phusion
PCR program (running time of just under 2 hours):
1. 98 degrees Celsius for 3 minutes
2. 98 degrees C for 15 seconds
3. 63 degrees C for 30 seconds
4. 72 degrees C for 1 minute
5. Go to step 2, repeat 30 times
6. 72 degrees C for 5 minutes
Ligation
Used the Ginkgo bioworks protocol
(Control: 2 uL of Circular PSB1C3 plasmid, 13 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase
P&C: 2 uL of Circular PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase
L&P: 2 uL of linear PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase
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