SLIM mutagenesis of large plasmids - (Jun/17/2011 )
I tried to mutate a large 16.5 kbp plasmid, according to QuikChange protocol. Polymerase was PfuTurbo. To some reactions I added DMSO and betaine to facilitate melting secondary structures. I tried different Ta, template amounts, primer design. Then I digested parental DNA with DpnI and tried to visualize mutated plasmid on a gel. There were no appropriate bands on a gel for all my trial mutagenesis reactions.
I heard about SLIM method adapted for large plasmids. Do anyone know this principle and details?
I think you should try transforming your mutation products, rather then trying to visualize them on a gel. You don't need a lot of molecules to get results you want, far fewer than you can visualize on a gel.
phage434 on Fri Jun 17 19:19:46 2011 said:
I think you should try transforming your mutation products, rather then trying to visualize them on a gel. You don't need a lot of molecules to get results you want, far fewer than you can visualize on a gel.
I'm currently doing that, but I was discouraged by visible absence of the mutated plasmid.
Hi Oleg,
May i know how long of base pairs you need to mutate? u mean all 16.5Kb? there's a kit if you targeting on a few mutations point in your plasmid...
Evanescence on Mon Jun 20 10:18:13 2011 said:
Hi Oleg,
May i know how long of base pairs you need to mutate? u mean all 16.5Kb? there's a kit if you targeting on a few mutations point in your plasmid...
I need to substitute 1 nucleotide at a time. Do you mean QuikChange II XL kit?
My friend was using fusion site directed mutagenesis from Finnzyme, how ever i noticed this only working for 10kb...I think the kit u were used still the best but my friend did suggst to choose best annealing temperature as a lil change will give effect to the product...so far 2 mutants obtained (unfortunately, the plasmid size is below than 10kb) which mean not suitable for you...if there's no problem to you would you send me your plasmid map together with complete restriction site? i am thinking to cut some point and doing some technical one without using kit...
i dont know what it will bring but we can think together
Oleg on Fri Jun 17 18:03:08 2011 said:
I tried to mutate a large 16.5 kbp plasmid, according to QuikChange protocol. Polymerase was PfuTurbo. To some reactions I added DMSO and betaine to facilitate melting secondary structures. I tried different Ta, template amounts, primer design. Then I digested parental DNA with DpnI and tried to visualize mutated plasmid on a gel. There were no appropriate bands on a gel for all my trial mutagenesis reactions.
I heard about SLIM method adapted for large plasmids. Do anyone know this principle and details?
I indicated incorrect DNA polymerase. Actually, my DNA polymerase was homemade Pfu, rather than PfuTurbo or PfuUltra. Probably this was the reason (but the size of my plasmid) of such inefficient mutagenesis.
phage434 on Fri Jun 17 19:19:46 2011 said:
I think you should try transforming your mutation products, rather then trying to visualize them on a gel. You don't need a lot of molecules to get results you want, far fewer than you can visualize on a gel.
Reporting intermediate results:
With Ta = 68C and partially overlapping primers (they overlapped each other at their 5'-termini, as described in (L. Zheng, U. Baumann and Jean-Louis Reymond. An efficient one-step site-directed and site-saturation. Nucleic Acids Research, 2004, Vol. 32, No. 14)) there were 12 colonies. Under the same conditions, but with completely overlapping primers there were 7 colonies. At lower Ta, there were no colonies.
I will report here sequencing results later.
I don't know whether PCR enhancers helped because I combined different samples. Future will show...
I did a lot of experiments with this mutagenesis, but all colonies I picked contained a plasmid of smaller size (about 7 kbp). How can it be explained?
I phenol-chloroform extracted my mutagenesis reactions, ethanol precipitated them and transformed bacteria with a half of DNA.