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Team:GeorgiaTech/Colony PCR Protocol
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===Materials=== | ===Materials=== | ||
*Prepare master mix: (Total volume '''50 µL''') | *Prepare master mix: (Total volume '''50 µL''') | ||
Latest revision as of 01:13, 28 September 2013
Main Page
Contents |
Colony PCR Protocol
Materials
- Prepare master mix: (Total volume 50 µL)
- 5X Rxn Buffer 10 µL
- Oligo 1 (5µM) 7.5 µL
- Oligo 2 (5µM) 7.5 µL
- dNTP mix 1 µL
- DMSO 1 µL
- ddH2O 22 µL
- Phusion 0.5 µL
- NOTE: you will need 10µl of the master mix per colony, plus for positive and negative controls. Adjust volumes appropriately.
- Oligos 1 and 2 are the primers for the BioBrick part, VF2 and VR. Make sure to dilute the concentration to 5 µM to prepare the appropriate amounts.
To prepare the cells
- If you have a liquid culture:
- Prepare as many PCR tubes as you have cultures you want to amplify. Add 100 µL of ddH2O to each of those PCR tubes.
- Take 3 µL from each culture you want to PCR amplify and add to the PCR tubes (make sure to properly label each tube).
- If you have plated colonies:
- Prepare a sterile environment (wipe down counter with ethanol, get out a bunsen burner and light with a striker).
- Sterilize metal innoculating loop with bunsen burner and scoop up one colony and mix into the appropriate PCR tube.
Procedure
- Prepare connected PCR tubes (one for each colony, one for positive control, and one for negative control)
- Add 10 µL master mix into each tube
- Immediately cap the negative control to prevent cross-contamination.
- Add 1 µL of diluted cell culture to its corresponding PCR tube (make sure to label everything)
- Use a positive control that you know will work and has a known part
- Close all caps and place in PCR Machine
- Run PCR program COLONPCR_Protocol
- Once this is complete, you will be able to analyze these colonies using gel electrophoresis and determine which colonies have the appropriate parts.
