I have performed transformation of my gene into Pichia to express my desired recombinant protein. Now that I have successfully transformed, I would like to know if my gene has been integrated into the genome of Pichia through homologous recombination? For this reason, I would like to extract the genomic DNA of the Pichia so that I can confirm the presence of my gene of interest. What is the best, cheapest and most reliable method of liberating the DNA?
Depends on how you want to confirm the presence of that gene. If by PCR, I usually resuspend part of a colony into 20 uL of 20 mM NaOH, heat for 45 min. at 98oC, and use 1 uL of the resulting supernatant for PCR. If you intend to do Southern blotting, then there is a recipe in the manual for the Pichia multicopy kit (from Invitrogen).
For routine PCR, you can make a slightly dense suspension of a Pichia colony in 100-200 ul of water, heat the suspension for 5 min at a temperature near to boiling (
Hi Dr. Alejandro and Dr. Tolstorukov,
Thanks for your suggestions. I tried both your methods today and have successfully liberated the gDNA from Pichia. Thank you a bunch for your invaluable input on this matter.
- Badges
- Science method