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It helps to remember that PCR does have an upper limit of total double-stranded DNA content (regardless of its molarity!) after which it does not work any more (due to the competition of the polymerase for non-specific dsDNA versus primer-substrate pairs). Therefore the theoretical limits on this form of cloning are imposed by the molarity of two DNA fragments, the fidelity/processivity of your PCR enzyme, and the quality of the reaction mix. In practice it helps to also consider the frequency of PCR errors as well as certain other factors. I've done this sort of cloning with ~4 KB inserts, however the use of such long inserts required optimization of conditions and was not 100% successful (unlike the relatively simple insertion of 0.5-1.5 KB). With long inserts it is *critical* to have highly purified and highly homogenous starter DNA as well as (individually selectable) a correct template/insert ratio. Artem -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Raji Edayathumangalam Sent: Monday, December 01, 2008 3:54 PM To: [log in to unmask] Subject: [ccp4bb] Quikchange cloning: Insert length Hi Folks, Sorry for the non-xtallo posting. I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also generate the initial PCR product. I plan to proceed with my insert which is ~ 2kb and am curious to get some feedback if you have successfully cloned inserts > 1.5kb using the above strategy. Many thanks. Raji