I am trying to amply a 1400bp gene.. My expected fragments which are 900 and 500 were achieved. I joined the two fragments and i got 1400 as expected. This is my mutation desire CAG to GAG.
But my sequencing result reveled it still wildtype(CAG) and not GAG? I use phusion polymerase my mutagenesis?
Any suggestion what will be the cause of this problem... Because if it is contamination how come it will happen if i gel clean the two fragments.
Hi, do you use an empty vector for the last step (overlap extension PCR with joined fragments)? So you are not using the vector with wild type version of the gene?
Did you sequence the joined fragments? If the joined fragments carry the mutation the problem lies somewhere downstream. If you don't see the mutation the problem is the first PCRs. Did you double check the primer?
Hi there Daniel!!!,
Irrespectively of the DNA template (plasmid or cDNA via reverse transcription of mRNA), you need to design two extreme primers (A -Fw- and D-Rv-) for the 1400 bp sequence and two primers somewhere in the middle of the 1400 bp sequence, both containing the desired mutation (B -Rv- and C -Fw-). B should be (totally or partially) the reverse complement of C. In any case, if the mutation is within the complementary region between B and C, both primers must contain the desired mutation.
Combine A/B and C/D primers in two separate PCR reactions. Check products by minigel and gel-purify the 500 and 900 bp fragments.
Combine A/D primers with the 500 and 900 bp purified fragments in a second PCR. If you get a 1400 bp product, there is no doubt that it will contain the desired mutation.
Does the product consist of a full gene or are you going to replace the PCR product with the wild sequence already cloned in a plasmid?. In that later case, make sure that there are unique restriction sites within the 1400 bp sequence and flanking the mutation(s) .
See attached file with a design example.
Hi my suggestion would be
design 4 primers two extreme ends 1 and 4...and 2 internal primers 2 and 3 overlapping only partially where the mutation is and both primers should carry the mutation.
now amplify the 2 regions 500(using primer 1 and 2) and 900 using(3 and 4). to fuse both of them use forward of first primer 1 and reverse of second primer 4 and amplify.
Hope this helps
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