Blackburn:Yeast Colony PCR: Difference between revisions

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Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0|Blackburn Lab:  Quick and Easy Yeast Colony PCR v2.0]]
==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.
Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0|Blackburn Lab:  Quick and Easy Yeast Colony PCR v2.0]]


==Materials==
==Materials==
List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.


*supply 1 (i.e. tubes of a certain size? spreaders?)
*Standard PCR machine, tubes
*reagent 1
*[http://www.qiagen.com/Products/Pcr/TaqSystem/TaqDnaPolymerase.aspx Qiagen Taq Polymerase Kit with Q-solution]
*X μL reagent 2
*A small yeast colony
**component A (reagent 2 is made up of multiple components)
*0.02M NaOH (3uL per reaction)
**component B
*equipment 1
*equipment 2


==Procedure==
==Procedure==
#Step 1
#Step 2
#*Step 2 has some additional information that goes with it.  i.e. Keep at 4°C.
#Step 3
##Step 3 has multiple sub-steps within it.
##Enumerate each of those.


==Notes==
===Yeast Cell Lysis===
#List troubleshooting tips here.   
#Aliquot 3uL of 0.02M NaOH into PCR tubes.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
#Anecdotal observations that might be of use to others can also be posted here.   
#*If the solution is cloudy, you've added enough cells.
#[optional] Quick-freeze the cells in liquid nitrogen
#*I normally skip this step.  Works just fine.
#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===


Please sign your name to your note by adding <font face="courier"><nowiki>('''~~~~''')</nowiki></font> to the end of your tip.
#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
#Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
#Run the following PCR cycle:
##5 min at 94C
##30 cycles of:
###30 sec at 94C
###30 sec at 55C (or appropriate annealing temperature)
###1 min/kbp at 72C
##10 min at 72C


==References==
==Notes==
'''Relevant papers and books'''
*Note that we use 0.5uL of 100uM primer, which is about ten-times more than standard PCR protocols.
<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
*The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
<biblio>
*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
#Goldbeter-PNAS-1981 pmid=6947258
*The expected PCR product should be as short as possible.  Anything less than 1kbp can be easily amplified.
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
</biblio>


==Contact==
==Contact==
*Who has experience with this protocol?
[[Special:Emailuser/Tetm2002|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])

Latest revision as of 18:13, 20 March 2009

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

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)

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