Blackburn:Yeast Colony PCR: Difference between revisions
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==Procedure== | ==Procedure== | ||
# | |||
# | ===Yeast Cell Lysis=== | ||
#* | #Aliquot 3uL of 0.02M NaOH into PCR tubes. | ||
# | #Using a sterile pipette tip, pick a small colony and resuspend in NaOH. | ||
## | #*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 sometime. Keep on ice or freeze for longer. | |||
===PCR=== | |||
#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 | |||
==Notes== | ==Notes== | ||
Revision as of 21:19, 11 June 2006
Overview
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
Materials
- Standard PCR machine, tubes
- Qiagen Taq Polymerase Kit with Q-solution
- A small yeast colony
- 0.02M NaOH (3uL per reaction)
Procedure
Yeast Cell Lysis
- Aliquot 3uL of 0.02M NaOH into PCR tubes.
- Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
- 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 sometime. Keep on ice or freeze for longer.
PCR
- 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
Notes
- 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 end of your tip.
References
Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
- ISBN:0879697164
Contact
- Who has experience with this protocol?
