McClean: Colony PCR (Yeast)

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Revision as of 11:25, 4 October 2011 by Megan N McClean (Talk | contribs)
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Contents

Overview

Our lab's version of yeast colony PCR, adapted from the Botstein Lab's protocol. Generally, we use this protocol for checking transformations (ie, to check that a drug marker or fluorescent protein has inserted into the genome correctly) or for PCRing up a piece of DNA from the genome to send for sequencing.

Materials

  • HotMaster Taq Polymerase
  • 10x HotMaster Buffer with Mg2+
    • The polymerase and buffer come in the 5 Prime kit FP220320 ordered from Fisher
  • 10mM dNTPs
  • Forward primer (10μM)
  • Reverse primer (10μM)
  • Sterile H2O

Protocol

Add approximately 0.6μL of cells (tiny amount) with the tip of a sterile toothpick into the bottom of PCR tubes or plate. Once you've put cells into the PCR vessel, put the end of the toothpick into ~100μL sterile YPD in either an eppendorf or the well of a culture plate. You will use this to inoculate an overnight culture if your colony PCR works. Keep the eppendorfs or culture plate at either room temperature or 30°C while you run the PCR, either is fine.

Microwave cells in the PCR tube/plate for 1min (2X). Put microwaved cells on ice.


Add the reaction mix (described below) to the PCR tube/plate. It is recommended to make up a master mix if you are doing multiple colonies. Put the PCR tubes/plate into the thermocycler and run the Colony PCR program described below.

PCR Reaction Mix

Reagent Volume
10x HotMaster Taq Buffer with Mg2+ 5μL
10mM dNTP mix 1μL
10μM Primer 1 1μL
10μM Primer 2 1μL
HotMaster Taq DNA polymerase 0.5μL
Sterile Water 40.5μL
Total Volume 50μL

PCR Program

Run on PCR on Colony PCR program (MeganColonyPCR):

  • 95°C 4min
  • For the following steps, reduce the temperature ‐1°C each cycle and cycle 30x's
    • 94°C 1min
    • 65°C 1min
    • 68°C 2min
  • For the following steps, cycle 30x's
    • 94°C 30sec
    • 50°C 30sec
    • 72°C 1min
  • 72°C 5min
  • 4°C Forever
    • Please don't leave the thermocycler running 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature). Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. 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.

References

Botstein Lab protocols: http://www.princeton.edu/genomics/botstein/protocols/colony_PCR.htm

Contact

Megan N McClean

or instead, discuss this protocol.


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