LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION
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Repeated freeze-thawing of primers will reduce the efficiency of this protocol. Make small working stock aliquots of primer and dispose of after 2-3 freeze-thaw cycles.
dNTPs
Prepare individual stocks at a concentration of 10 mM in sterile water.
Ensure dNTPs are fresh – the freeze-thawing rule applies to these too.
deaza GTP
This is supplied by Boehringer Mannheim at a concentration of 10 mM.
Sub-aliquot this to minimise freeze-thawing.
Taq Polymerase-
Use the Expand “kit” from Boehringer Mannheim (Catalogue number 1681842). This comes with the appropriate buffer and Taq polymerase mix for optimal long template extension.
The mix includes a proof-reading Taq that will degrade single-stranded DNA, including primers. To prevent degradation, the Taq mix (master mix 1 - see below) needs to be kept separate from the primer containing mix (master mix 2) until immediately before thermal cycling.
PCR Buffer 2 (x10)
This is supplied with the Expand kit and is used at a final concentration of 1x in the reaction mix
An alternative to the Boehringer kit is the DyNAZyme EXT kit manufactured by Finnzymes. This kit is cheaper but you may encounter problems when using the supplied buffer. The DyNAZyme polymerase mix with Boehringer buffer 2 seems to work very well!
Sample Loading Buffer (5x)
4.8 ml glycerol
0.025g bromophenol blue (0.25%)
0.2 ml 20% SDS
5 ml 10x TBE
10x TBE
108g Tris
55g boric acid
40 ml 0.5M EDTA, pH 8.0
to 1l with distilled water.
1kb BASE PAIR LADDERS (Gibco BRL)
