I need a protocol of colony PCR for confirmation of inserts after the Pichia pastoris electroporation. I performed a protocol, however, it didn't amplified any bands. So, I would like to compare to another one. Does somebody has any protocol (from collect colony until the pcr mix) for colony PCR of P. pastoris transformants?
Pichia pastoris cell wall may hinder a direct colony PCR. You will have to use lyticase to dissolve cell wall. A protocol is available in the invitrogen manual
http://tools.lifetechnologies.com/content/sfs/manuals/pich_man.pdf
Try this protocol
http://www.sbs.utexas.edu/jayaram/jayaramlab_files/newlabwebpage_files/Protocols/Yeast%20Colony%20PCR%20protocol.pdf
Thanks guys! I did a protocol with SDS 0.2%! I am waiting for the results on gel...
I have have done a lot of colony PCR and it work,however I use a PCR ready mix (13ul) primer1(1ul) primer2 (2ul) and just add colony to it.The PCR program depent on what your primers are(Annealing).I can check cat no.of this ready mix and update you.
As mentioned already Pichia cells are tougher than E. coli to lyse. Only use fresh colonies (preferably not greater than 24hrs, definitely less than 36). I also found that picking and adding colony straight to pcr mix and then putting in thermocycyler usually didn't work. Instead I pick and add cells to 8ul water in a pcr tube then microwave full power or boil for 4-5mins (if microwave don't use a rack to hold tubes - the rack seems to shield tube from microwaves). Then add 5ul of this to your pcr mix. Works every time!
see link for protocol http://enzyme.ucd.ie/main/index.php/PCR#Pichia_Colony_PCR
I used this very simple protocol and it worked very well:
- Resuspend each colony in 20ul of sterile water
- take 10ul,add 5U lyticase and incubate 10min at 30 C then freeze at -80C for 10min
- Prepare your PCR mixture using 5ul of the above lysate as DNA source.
Fernada,
did you find the solution? how did you check your colonies? i have the same problem!
Thanks to all! I got a solution for this problem!!
Eleni, I tried 3 differents ways for colony PCR:
1) Using a toothpick, pickup the colony and add to the PCR mix, or
2) Treating the colony with SDS 0,2% and add Triton (I forgot the concentration, I think it is 10%) in the PCR mix, or
3) Boiling the colony in water for 5 minutes and using 1 uL per reaction!
The second and third worked very well!! I prefered the last one, because if the SDS is in a wrong concentration, the reaction won't work.
If you need the exactly protocol, tell me and I will send to you!
Good luck!!
After trying several methods, I figured out a simple way out.
I prepared a cell lysate by boiling single colonies in 20ul of sterile water at 95°C/5min.
No lyticase added.
Used 1ul of the lysate in 10ul of PCR mix,
PCR program 30 cycles of 95,55,72.
PCR worked perfectly in "Ampliqon Taq DNA polymerase 2x PCR mix" Cat.No. A180301
The same lysate template gives inconsistant results when Taq polymerase from different manufacturers were used.
Good luck.
Hi. I think accurate results can be obtained from the genomic DNA extracted from some your transformants. So may look for the protocol for extraction of yeast genomic DNA
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