Hi all,
Recently I've been trying to test different genomic DNA extraction from yeast to quickly check genotypes of my transformed yeast by colony pcr. Since the traditional gDNA extraction took really long time (long incubation of PK...) I found two literature that describe relatively quick and dirty way to release nucleic acid and subject the samples to PCR.
GC preps:
https://www.ncbi.nlm.nih.gov/pubmed/27240644
Grab n Bust method:
http://www.openwetware.org/wiki/McClean:_Genomic_DNA_Prep_(Bust_'n'_Grab_Protocol)
I tried both methods, but for GC preps (a relatively new method, and few ppl gave feedback about it) I couldn't get much PCR product.
For Bust n Grab method, I succeeded in isolating DNA and get PCR works in liquid culture sample but not colony.
I understand that these methods are really quick and dirty things and might not work well if I wanna apply it to Southern blot or any Enzymatic digestion, but I think it will be useful at the stage of narrowing down my candidates of mutants.
Just wondering if anyone had tried different gDNA isolation methods and if there's any possible modification I can make to them.
Thanks!!!!
Peggy
Vortexing yeast suspension with glass beads is an old method to break cell wall integrity (relatively tough) but results can be variable depending on cell density, relative proportion of glass beads to cells and volumes, and finally the time and temperature. Not enough and you will have poor yield, too much and the DNA will be severely sheared.
I would suggest making a stock of Zymolyase and aliquot 50-100microL of this solution into microcentrifuge tube, add cells from a colony to a tume, incubate for a finite time (you will have to test this step in your own hands; 10-20min should do) - then add glass beads and vortex, cells will readily and reliably open up with a brief vortexing (again, test it in your hands to see how long it takes to get optimum results).
Once you have optimized this method on a few colonies, the method will work well for your strain of yeast (and similar strains).
I used lyticase, followed by phenol:chloroform extraction and ethanol precipitation. Its been working pretty well. Here is my protocol
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology