Amplifying *large* DNA...??

nigel at codon1.berkeley.edu nigel at codon1.berkeley.edu
Wed Nov 13 20:19:12 EST 1991


Greetings Netsters

I've got another question for y'all......

Does anyone out there have a TRIED and true recipe for amplifying large [>4kb] 
peices of DNA, using PCR, that they would care to share?

I just failed to amplifiy a 4.9kb fragment using the "vector PCR" protocol
that was published in last months BioTechniques [p446-452]:
		- 100uL rxn vol.
		- 200uM dNTP 
		- 1.5mM MgCl2
		- 50mM KCl
		- 10mM Tris  [pH 8.3]
		- 0.01% gelatin
		- 25 pMoles each primer                
		- 2.5 U AmpliTaq       
		- 1-10 ng template   

	- 2 min @ 94 C; 2 min @ 52 C; 5 min @ 74 C   for 30 rounds.

I actually used 50 pMoles of a single primer that should prime at either end of
my insert and an unknown amount of template [~ 200ng].

I'm about to start playing with the conditions, and any clues would be much
appreciated.

Cheers!

-n

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