PCR for 6-7 kb

Shiao Y. Wang sywang at whale.st.usm.edu
Fri Apr 1 12:43:41 EST 1994


Jeffrey Vance (jmv at galactose.mc.duke.edu) wrote:
: 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***

Can you provide some details or point the direction for the Tricine-KCl
buffer and the "shrimp method"? Thanks.




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