Homemade PCR gems

gstuart at ac.dal.ca gstuart at ac.dal.ca
Tue Jun 9 13:30:18 EST 1992


In article <920604105949.21e01083 at ntet.nctr.fda.gov>, WMELCHIOR at NTET.NCTR.FDA.GOV (Bill Melchior, NCTR/FDA) writes:
> A couple of months ago, the following was posted:
> 
>>From:	MX%"ageorge at mbio.med.upenn.edu"  1-APR-1992 12:18:11.57
>>Subj:	Homemade PCR gems
>>
>>Has anyone perfected a method for producing "home-grown" wax pellets
>>for use in "hot-start" PCR?  We have been playing around with various
>>substances and have had reasonable success with Sigma Paraplast.  We
>>heat a few grams of the material in a few milliliters of sterile water
>>in the microwave until melted.  Then use a "sawed off" pasteur pipette
>>to drop the molten paraplast into cold sterile water to form the
>>"gems".  We are working on getting the size standardized.  The cost
>>per pellet is quite low.
>>	Any other ideas??
>>Al George
> 
> I'd like to know whether Al or anyone else has made any progress on the
> techniques since that posting.  Thanks, Bill
I have a homemade method that might help. Buy some paraffin wax
(Parowax) - the kind that is used for sealing bottled preserves. 
For a 100 microlitre PCR rxn add primers, 1/2 req'd amt. 10X 
buffer, dNTP's, and H2O to 50 microlitres. Then, on a piece of
clean parafilm and with a clean blade, cut off a small chunk of 
Parowax, and add it to the tube. Cap, and put in a heatblock @
70-80C. The wax will melt in a minute or two, and excess wax can 
be pipetted off easily with a P200 Pipetman. Remove the tube to the bench; it will harden within 1-2 min. Then add, on top of the wax, 50 micro-
litres containing the other 1/2 req'd. amt. 10X buffer, the Taq  DNA polymerase, and your DNA. 
In the PCR machine the wax will start to melt @ ~50C and will be completely melted at ~70C. 
the PCR rxn.
One should test the wax alone to ensure it (minus DNA) doesn't give PCR product. 
Let me know if it works/helps!



More information about the Methods mailing list