Stanford/BIOE44:Module 5:Day3: Difference between revisions
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===I0500 troubleshooting=== | |||
Revision as of 00:41, 25 May 2010
M5: Day 2 - Continue Building
Freezer Stocks of Synthesized Parts
Materials
- 5mL overnight culture
- sterile 60% glycerol
- 3 cryovials, labelled
Procedure
- Label your cryovials: part name, plasmid, date, your initials.
- Add 500ul of sterile 60% glycerol to each of your cryovials.
- Add 1.5mL of overnight culture to each cryovial. (Its easiest to use a 5ml pipette, take up 4.5mL of culture then drop 1.5mL into each tube)
- Invert tubes several times to mix.
- Put in freezer box in -80C freezer. (Usually one tube is kept as the working stock and the other two are kept as back ups in a separate freezer.)
PCR amplifying your parts
Your primers have arrived! This is the packing list that comes with the primers when they are delievered. It has a lot of useful information.
We will use this polymerase: Pfx50 DNA polyermase It is higher fidelity than TAQ (which means it makes less mistakes).
PCR mix
| Component | Volume | Final Conc. |
| 10X Pfx50 buffer | 5ul | 1X |
| 10mM dNTPs | 1.5ul | 300uM |
| fwd primer | 300nM | |
| rev primer | 300nM | |
| Template | 1-100pg | |
| Pfx50 polymerase | 1ul | |
| Water | ||
| Total volume | 50ul |
PCR Protocol
(taken from Pfx50 manual)
| Cycles | Temp | Time |
| 1x | 94C | 2 min |
| 35x | 94C | 15s |
| 60-68C (Tm primers - 2C) | 30s | |
| 68C | 30s per kb | |
| 1x | 68C | 5 min |
| 1x | 15C | forever |
