Blackburn:Yeast Colony PCR

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==Procedure==
==Procedure==
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#Step 1
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#Step 2
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===Yeast Cell Lysis===
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#*Step 2 has some additional information that goes with iti.e. Keep at 4°C.
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#Aliquot 3uL of 0.02M NaOH into PCR tubes.
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#Step 3
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#Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
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##Step 3 has multiple sub-steps within it.
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#*If the solution is cloudy, you've added enough cells.
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##Enumerate each of those.
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#[optional] Quick-freeze the cells in liquid nitrogen
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#*I normally skip this stepWorks just fine.
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#Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
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#*In the mean time, prepare the master mix for the PCR reaction.
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#*The boiled samples are stable at room temp for sometime.  Keep on ice or freeze for longer.
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===PCR===
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#Prepare the master mix solution containing:
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#*5uL 5X Q-solution
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#*2.5uL 10X PCR Buffer
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#*0.5uL dNTPs (10mM each)
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#*0.5uL foward primer (100uM)
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#*0.5uL reverse primer (100uM)
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#*0.25uL Taq
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#*12.75uL ddH2O
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#Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
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#Run the following PCR cycle:
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##5 min at 94C
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##30 cycles of:
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###30 sec at 94C
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###30 sec at 55C (or appropriate annealing temperature)
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###1 min/kbp at 72C
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##10 min at 72C
==Notes==
==Notes==

Revision as of 00:19, 12 June 2006

Contents

Overview

This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.

Materials

Procedure

Yeast Cell Lysis

  1. Aliquot 3uL of 0.02M NaOH into PCR tubes.
  2. Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
    • If the solution is cloudy, you've added enough cells.
  3. [optional] Quick-freeze the cells in liquid nitrogen
    • I normally skip this step. Works just fine.
  4. Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
    • In the mean time, prepare the master mix for the PCR reaction.
    • The boiled samples are stable at room temp for sometime. Keep on ice or freeze for longer.

PCR

  1. Prepare the master mix solution containing:
    • 5uL 5X Q-solution
    • 2.5uL 10X PCR Buffer
    • 0.5uL dNTPs (10mM each)
    • 0.5uL foward primer (100uM)
    • 0.5uL reverse primer (100uM)
    • 0.25uL Taq
    • 12.75uL ddH2O
  2. Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
  3. Run the following PCR cycle:
    1. 5 min at 94C
    2. 30 cycles of:
      1. 30 sec at 94C
      2. 30 sec at 55C (or appropriate annealing temperature)
      3. 1 min/kbp at 72C
    3. 10 min at 72C

Notes

  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 end of your tip.

References

Relevant papers and books

  1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Jacob-JMB-1961]
  3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed HubMed

Contact

  • Who has experience with this protocol?
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