McClean:Colony PCR (Quick Load)
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Overview
Fast colony PCR protocol for quickly screening many yeast colonies. This protocol uses OneTaq Quick-Load 2X Master Mix (NEB #M0486).
- Cheap (~$0.40/50μl rxn)
- Fast to set up
- Scales down well to small volumes. I've done colony PCR with 25μl, 10μl, and 5μl volumes and saw no difference in efficiency.
- Load onto gel directly after thermocycling -- the master mix contains 2 dyes, Xylene Cyanol FF and Tartrazine, which run at ~4kb and ~10bp respectively on a 1% agarose gel.
- Not hot start -- set it up on ice and preheat the thermocycler to 94°C.
Materials
- OneTaq Quick-Load 2X Master Mix with Standard Buffer (NEB #M0486)
- H2O
- Primers
- Colonies
- Sterile toothpicks
Procedure
- Start the thermocycler protocol (see below) to allow the block to preheat to 94°C.
- In a PCR tube, mix the components on ice. For a large screen, set up a master mix then aliquot into PCR tubes.
- For a 25μl reaction:
- 12.5 μl OneTaq master mix
- 0.5 μl Primer 1 (200 nM final concentration)
- 0.5 μl Primer 2 (200 nM final concentration)
- 11.5 μl H2O
- For a 25μl reaction:
- Using a sterile toothpick, pick a colony and gently dip into the reaction until it becomes noticeably cloudy. Don't overdo it, faint cloudiness is enough.
- Flick tubes gently to mix and collect with a quick spin
- Place cells in preheated thermocycler and press 'Enter' to continue protocol
- After thermocycling, load 5 μl directly onto agarose gel for visualization
Temp (°C) | Time | Purpose | |
---|---|---|---|
94 | HOLD | preheat | |
94 | 5 min | cell lysis | |
94 | 15 sec | 30X | denaturation |
Ta | 15 sec | annealing | |
68 | 1 min/kb | extension | |
68 | 5 min | final extension | |
4 | 10 min | stop reaction |
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
*Taylor D. Scott (talk) 15:33, 20 August 2019 (PDT): Use the NEB Tm calculator to calculate the annealing temp.
*Taylor D. Scott (talk) 15:33, 20 August 2019 (PDT): I have not tried this with E. coli but it probably will work.
References
Relevant papers and books
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.