MilliporeSigma
Expand Long Template PCR system
11681834001
I was trying to clone 18 Kb PCR product (PCR product is a 12 gene operon) from Bacillus subtilis into E. coli. Tried several PCR amplification kit but found htat Sigma's Expand Long Template PCR system performed very well to amplify 18 kb operon without any errors.
Generation of Long PCR fragment for various cloning purposes
250 ng of Bacillus subtilis genomic DNA
Protocol was performed according to manufacturer's instructions which came with PCR amplification kit. 50 microliter reactions were setup with 250 ng Bacillus subtilis DNA as template for amplification. Used Buffer 3 for amplification which contains 2.75 mM MgCl2 for boost DNA polymerases activity. In thermal cycling, 2 step extension was performed according to kit's instructions.
Kit comes with different Buffer systems. Use accordingly w.r.t length of fragment to be generated. Pre-heat Buffers to 50 degree C.
50 microliter PCR reactions with Expand Long Template PCR system using Buffer 3 component yielded 4 microgram of 18 Kb PCR product. Quality was checked by nanodrop measurements. PCR fragment was used to construct various plasmids for cloning.
N/A
A…Learn More
Easy to follow protocol. Best of long template PCR.
None
Need to standardize PCR with different buffer components which come with kit to get ideal amplification of your target DNA. Also sometimes need to perform gradient PCR.