Hello every one. I am studying the role of phosphorylation of TFII-I in neural precursor cells and Dopamine committed cells. I have done point mutation at 248 phospho site i.e change the codon (from tyrosine to Phenylalanine). I have performed PCR 1 to ampfiy gene with desired mutation and fragment 2 via using PCR 2. Both the gene fragments have a common sequence with a point mutation. Now I have to combine both the 2 fragments to get a whole gene using PCR. Kindly suggest me How I can combine the 2 fragments and which type of PCR we can use for this study?
Hi Muhammad,
you have 2 overlapping fragments, right?
In that case, you can dilute both PCR products (higher dilution is better than lower), combine together and add PCR mix with two primers, spanning the whole region (ie left primer of PCR product 1 and right product of PCR product 2). and run PCR. Your original PCR products (after denaturation) would serve as primers and once the combined product is obtained, PCR should run without any troubles. Try different cycles of PCR - might be cycle number dependent.
You can also try Gibson assembly for joining, and then amplify your fragment using PCR.
Martin
Dear Muhammad ,
For joining the two fragments of your interest first you have to know the exact sequence (by using sequencing) your isolate.Then try to findout the probable restriction sites that both the fragments having. Then cutted both the fragment using the above restriction enzyme followed by ligation using T4 DNA Ligase. But before going to restricted digestion u must be pretty sure about the restriction enzyme and its compatibility with each other.
Hi Muhammad,
you have 2 overlapping fragments, right?
In that case, you can dilute both PCR products (higher dilution is better than lower), combine together and add PCR mix with two primers, spanning the whole region (ie left primer of PCR product 1 and right product of PCR product 2). and run PCR. Your original PCR products (after denaturation) would serve as primers and once the combined product is obtained, PCR should run without any troubles. Try different cycles of PCR - might be cycle number dependent.
You can also try Gibson assembly for joining, and then amplify your fragment using PCR.
Martin
Overlaping PCR is best option for you try to make primer from fragment and one primer from other fragment then amplify it. OR Gibson Assembly is most precise if you have problem of large fragment.
Thanks
May I know the reason why you want to combine the two fragment?
If your purpose is sequencing, you can do sequencing separately and generate a contig using software as your PCR product contain overlapping reason.
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