Smallest insert in PCR - (Jan/10/2012 )
I am doing small gene insert in my gene of interest (450bp). My insert are very small length 6bp and 9 bp.
Is this insert could distinguish in Agarose gel when compare with non insert PCR product?
No. You might (should) be able to distinguish them using sequencing acrylamide gels and/or sequencing
Or fragmentation analysis (usually the same machine as sequencer). That would be most suitable IMO, since it can even quantify them. You would need one of your regular PCR primers to be re-synthethised with a fluorescent dye though.
You can pcr amplify only a small portion of your gene, surrounding the insertion. This will give fragments with a larger relative difference in length. Then, running these on a 20% TBE gel, you should be easily able to determine the insert presence compared to a gene without the insert.
Thanks to all.
to be absolutely sure, i will sequence it.