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| <h2>Making Glycerol Stock </h2> | | <h2>Making Glycerol Stock </h2> |
- | Use flasks and bottles before graduated cylinders (they take forever to mix!)
| + | To make 400 mL of 10% glycerol you will need: |
- | 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
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- | Don’t autoclave!
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- | <h2>Making ddH2O</h2>
| + | -40 mL glycerol |
- | Use the small bottles. Autoclave water by total volume.
| + | -360 mL of MilliQ water |
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- | <h2>Autoclaving </h2>
| + | To make 400 mL of 20% glycerol you will need: |
- | 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.
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- | <h2>PCR Troubleshooting</h2>
| + | -80 mL glycerol |
- | 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
| + | -320 mL of MilliQ water |
- | Buffers tested: GC + DMSO, HF + DMSO, GC, buffer G supermix + DMSO (3 tubes of each)
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- | 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.
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- | Master Mix: 10 uL dNTP’s, 2 uL forward primers, 2 uL reverse primers, 2 uL template DNA, 1 uL phusion
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- | 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)
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- | 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)
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- | 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)
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- | 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
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- | PCR program:
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- | 1. 98 degrees Celsius for 3 minutes
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- | 2. 98 degrees C for 15 seconds
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- | 3. Temperature gradient 48-55-63 degrees for 30 seconds
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- | 4. 72 degrees C for 1 minute
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- | 5. Go to step 2, repeat 30 times
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- | 6. 72 degrees C for 5 minutes
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- | Gel results: Only buffer G +DMSO works and it works at all temperatures.
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| + | 1. Use flasks and bottles before graduated cylinders (they take forever to mix!) <br/><br/> |
| + | 2. Need a stir plate and a large stir bar <br/><br/> |
| + | 3. Stir until mixture is homogeneous <br/><br/> |
| + | 4. Don’t autoclave! <br/><br/> |
| | | |
- | <h2>Optomized PCR</h2> | + | <h2>Making ddH2O</h2> |
- | Worked using buffer G +DMSO supermix at 63o C.
| + | - Use the small bottles. <br/><br/> |
- | 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
| + | - Autoclave water by total volume. <br/><br/> |
- | 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
| + | |
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| + | <h2>Autoclaving </h2> |
| + | - Robert is the source of official help on all things about the Autoclave. <br/><br/> |
| + | 1. Look at the Betastar for settings. <br/><br/> |
| + | 2. Go by materials and total volume. <br/><br/> |
| + | Use the autoclave tape and make sure it has changed colour by the end of the cycle. <br/><br/> |
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| <h2>Ligation </h2> | | <h2>Ligation </h2> |