Blackburn:Yeast Colony PCR

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Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0|Blackburn Lab:  Quick and Easy Yeast Colony PCR v2.0]]
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==Overview==
==Overview==
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
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 +
Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0|Blackburn Lab:  Quick and Easy Yeast Colony PCR v2.0]]
==Materials==
==Materials==
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*Standard PCR machine, tubes*Qiagen Taq with Q-solution
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*Standard PCR machine, tubes
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*[http://www1.qiagen.com/Products/Pcr/TaqSystem/TaqDnaPolymerase.aspx|Qiagen Taq Polymerase Kit with Q-solution]
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*[http://www.qiagen.com/Products/Pcr/TaqSystem/TaqDnaPolymerase.aspx Qiagen Taq Polymerase Kit with Q-solution]
*A small yeast colony
*A small yeast colony
*0.02M NaOH (3uL per reaction)
*0.02M NaOH (3uL per reaction)
==Procedure==
==Procedure==
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#Step 1
 
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#Step 2
 
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#*Step 2 has some additional information that goes with it.  i.e. Keep at 4°C.
 
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#Step 3
 
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##Step 3 has multiple sub-steps within it.
 
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##Enumerate each of those.
 
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==Notes==
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===Yeast Cell Lysis===
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#List troubleshooting tips here.   
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#Aliquot 3uL of 0.02M NaOH into PCR tubes.
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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#Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
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#Anecdotal observations that might be of use to others can also be posted here.   
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#*If the solution is cloudy, you've added enough cells.
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#[optional] Quick-freeze the cells in liquid nitrogen
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#*I normally skip this step.  Works 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 some time.  Keep on ice or freeze for longer.
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Please sign your name to your note by adding <font face="courier"><nowiki>('''~~~~''')</nowiki></font> to the end of your tip.
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===PCR===
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==References==
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#Prepare the master mix solution containing:
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'''Relevant papers and books'''
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#*5uL 5X Q-solution
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<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
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#*2.5uL 10X PCR Buffer
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<biblio>
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#*0.5uL dNTPs (10mM each)
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#Goldbeter-PNAS-1981 pmid=6947258
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#*0.5uL foward primer (100uM)
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#Jacob-JMB-1961 pmid=13718526
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#*0.5uL reverse primer (100uM)
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#Ptashne-Genetic-Switch isbn=0879697164
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#*0.25uL Taq
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</biblio>
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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
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==Notes==
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*Note that we use 0.5uL of 100uM primer, which is about ten-times more than standard PCR protocols.
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*The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
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*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
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*The expected PCR product should be as short as possible.  Anything less than 1kbp can be easily amplified.
==Contact==
==Contact==
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*Who has experience with this protocol?
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[[Special:Emailuser/Tetm2002|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])

Current revision

Updated Protocol: Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0

Contents

Overview

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

Updated Protocol: Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0

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 some time. 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

  • Note that we use 0.5uL of 100uM primer, which is about ten-times more than standard PCR protocols.
  • The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
  • For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
  • The expected PCR product should be as short as possible. Anything less than 1kbp can be easily amplified.

Contact

Tet (Blackburn Lab)

Retrieved from "http://www.openwetware.org/wiki/Blackburn:Yeast_Colony_PCR"
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