Re: [ccp4bb] Quikchange cloning: Insert length
Have you seen these papers: M Geiser, R Cebe, D Drewello, and R Schmitz. Integration of pcr fragments at any specific site within cloning vectors without the use of restriction enzymes and dna ligase. Biotechniques, 31(1):88–90, 2001. W Wang and B A Malcolm. Two-stage pcr protocol allowing introduction of multiple mutations, deletions and insertions using quikchange site-directed mutagenesis. Biotechniques, 26(4):680–682, 1999. If i recall correctly, Geiser el al inserted a 1kb fragment with a modified Quickchange method. On Mon, Dec 1, 2008 at 12:54 PM, Raji Edayathumangalam [EMAIL PROTECTED] wrote: 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 Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: [EMAIL PROTECTED] lab: 858-784-7758
Re: [ccp4bb] Quikchange cloning: Insert length
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. Here, the maximum we've tried was 3 kbp and it worked just as well as with the the smaller fragments. Most of the Quickchange cloning we did involved 1500-300 bp and no obvious differences in success rate vs size come to mind. Dima
Re: [ccp4bb] Quikchange cloning: Insert length
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:[EMAIL PROTECTED] On Behalf Of Raji Edayathumangalam Sent: Monday, December 01, 2008 3:54 PM To: CCP4BB@JISCMAIL.AC.UK 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
