PCR amplification of >6Kbp DNA - (Aug/24/2005 )
I need to subclone >6Kb DNA into plasmid for which I need atleast 300-500ng of the insert. The source of the DNA is phage, from which recovery of DNA is not very good. So I was wondering if amplifying the insert (>6Kb) using PCR will be a good idea? Has anybody amplified such big DNA? Any help is appreciated.
Long Range PCR (>5kb) is pickier than normal PCR and requires slightly larger primers (18-24bp) with slightly higher Tm (60-63C) but I have had repeated success with it, even from genomic DNA. I swear by the epicentre failsafe PCR kits (Dont worry I don't work for them or anything). The directions can be a bit deceiving by how simple they make it seem. Try the protocol they give you with the kit first and then try the gradient feature of your PCR machine with any premixes that give you even slightly hopeful results (such as schmearing) or with a random sampling of premixes (something like A, D, G, J, L). If you manage to get a faint band, try a temperature ramp (set the anneal temp for the first few cycles to be very strict and relax the anneal temp a few degrees after that) to get a stronger band.
Hope that helps.
Even larger primers(>30mer) work well to long-PCR, but don't forget to set set the Tm of two primers as equal as possible.
Also don't forget to use long-PCR specific Taq (I usually use LA Taq from TAKARA), and set the extension time longer than usual (1min per 1kb).
I amplified 8 kB fragment without difficulty.