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Site directed mutagenesis

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#1 kingswill



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Posted 03 December 2010 - 01:09 PM

I'm doing site directed mutagenesis to point mutate one bp using Phusion SDM kite from Finnzyme. I did the PCR and run a bit on gel to check. The product looks smaller than it should be and I'm a bit concerned now and would like to ask anybody here has experience in using this kit or SDM in general. Let me give some more details:

Source of template: miniprep plasmid
Primers: 5'phosphrylated and RP-HPLC purified (a must for this kit). double checked (in silico) they matched the regions I wanna mutate and don't cross-hybridize to any other region of the gene or in the backbone.
Expected product size (=size of the contruct): 6kb
PCR program: 98 10s, (98 10s, 72 3min30s) x 25, 72 10min (Tm is close to 72 (71.8), so the annealing and extension steps are combined. Extension time is based on the rate of 30s/kb)

After PCR I run the product on gel (1%). It appears around 4kb. I run alongside a linearized version of the same construct and it appear correctly at around 6kb. I have a control without Phu enzyme and there's nothing, which means that first template doesn't contribute to anything in the gel and second any bend in my sample should come from PCR amplification. I also have a positive control using a control plasmid provided by the kit which show a bend with correct size. This seems eliminating the possibility that anything eg salt and protein etc in the PCR reaction mix alter the migration pattern of the DNA in agarose gel.

I keep going to ligate the product and will transform them with a hope that the product is alright. However I drop my question here first in case I have to troubleshoot it later (fingercross I don't need to!)

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