Team:UIUC-Illinois/Notebook/Protocols/Bootcamp
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- | <center><h1> | + | <center><h1>Bootcamp Protocols</h1></center> |
+ | |||
+ | <h2>Making LB for plates</h2> | ||
+ | |||
+ | Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl | ||
+ | Add dry ingredients first. | ||
+ | To make 1 Liter of LB, use a 2L flask. Add 10 g Tryptone, 5 g Yeast Extract, 5 g NaCl, 1.5 g Agar (NOT agarose!). Then add 1 L of MilliQ water. | ||
+ | Autoclave by total volume. | ||
+ | Pour 25 mL on each plate (just enough to cover the bottom). | ||
+ | |||
+ | <h2>Making Liquid Media </h2> | ||
+ | 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 | ||
+ | |||
+ | <h2>Making Glycerol Stock </h2> | ||
+ | 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! | ||
+ | |||
+ | <h2>Making ddH2O</h2> | ||
+ | Use the small bottles. Autoclave water by total volume. | ||
+ | |||
+ | <h2>Autoclaving </h2> | ||
+ | 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. | ||
+ | |||
+ | <h2>PCR Troubleshooting</h2> | ||
+ | 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. | ||
+ | |||
+ | |||
+ | <h2>Optomized PCR</h2> | ||
+ | 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 | ||
+ | |||
+ | |||
+ | <h2>Ligation </h2> | ||
+ | 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 | ||
+ | |||
+ | |||
</div> | </div> | ||
Revision as of 18:48, 1 June 2012