PCR for 6-7 kb

[Jeffrey Vance]

Dan Diaz (bl275 at cleveland.Freenet.Edu) wrote:

: In a previous article, lukasz at wlheye.jsei.ucla.edu (Lukasz Salwinski) says:

: >I've got no problems with PCRing whole plasmids (pUC18 with
: >an insert) as large as 5.1kb from both minipreps and single
: >colonies. It only difference from the standard protocols
: >is I extended incubation time to 6 min (ie roughly 1min/kb)

Zgadzam sie! We do similar PCRs on human genomic DNAs with primers that
span exons. For products in this range, 2-6kB, we made our own Tricine-KCl
buffer (pH 8.8) and increased elongation cycles to 5 min. in an otherwise
standard setup. At the recent ASHG meeting, it was reported (& undoubtably
published) that whole lambda-clone inserts of >20kB can be produced using
Tricine buffer, glycerol (up to 10%), and longer (~10 min.) elongation
times. For the "long range PCR" (recently in Science), polymerases with
better processivity (eg Pfu, Vent) are substituted. 

Hey...does anyone know whether these long-range polymerases can be used
for sequencing? These PCR products sequence well, but I can't quite get
2kB from sequencing from both end primers.(-:..yes, we use the shrimp
method...:-)

*DB Loeb*
***DUMC Neurology***