Paul.xu: Created page with "http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html For PCR with low-complexity templates (e.g., plasmid and cosmid inserts)
50 microliter reaction in 1X Long P..."

2019-05-24T19:31:47Z

Created page with "http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html For PCR with low-complexity templates (e.g., plasmid and cosmid inserts) 50 microliter reaction in 1X Long P..."

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http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html

For PCR with low-complexity templates (e.g., plasmid and cosmid inserts) 50 microliter reaction in 1X Long PCR buffer (5X recipe below)

*2U to 5U Tth
*0.02 U Vent
*0.2 mM each dNTP
*20-40 pmoles each primer (400-800 nM)
*1.2 mM Mg(OAc)2
*10^5 -10^7 template molecules

For PCR with moderate-complexity templates (e.g., bacterial genomic DNA) 50 microliter reaction same as above except for the following change: 0.1 U Vent per rxn. This Vent concentration has not been completely optimized and more Vent might be better, but this concentration works well. For PCR with high-complexity templates (e.g., human genomic DNA) Others have informed us that conditions similar to those above work well. Long PCR Buffer In our hands tricine buffer works well with Tth but not as well with Taq. The pH is probably critical to the efficiency of amplification of long targets (1).

1X Long PCR Buffer

*85 mM KOAc
*25 mM Tricine pH 8.7 (adjust pH of Tricine stock solution with KOH)
*8% glycerol
*1% DMSO (1 to 4% works)
*1.2 mM Mg(OAc)2

A 5X buffer stock containing 6 mM Mg(OAc)2 (1.2 mM final concentration) and 5% DMSO (1% final concentration) can easily be made using 500 mM Mg(OAc)2, 1 M Tricine and 1 M KOAc stock solutions as follows:

*5X Long PCR Buffer
*425 mM KOAc
*125 mM Tricine pH 8.7 (adjust pH of Tricine stock solution with KOH)
*40% glycerol
*5% DMSO
*6.0 mM Mg(OAc)2
*10 ml 5X buffer
*4.25 ml 1M KOAc
*1.25 ml 1M Tricine, pH8.7 @ 25 degrees C (with KOH)
*4.00 ml glycerol
*0.50 ml DMSO
*120 ul 500 mM Mg(OAc)2
[[Category:Protocols]]

Long PCR - Revision history

Paul.xu: Created page with "http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html For PCR with low-complexity templates (e.g., plasmid and cosmid inserts)50 microliter reaction in 1X Long P..."

Paul.xu: Created page with "http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html For PCR with low-complexity templates (e.g., plasmid and cosmid inserts)
50 microliter reaction in 1X Long P..."