I have had some trouble to amplify a gene of 500 pb using as a template cDNA from Mouse. there is many fragments in the amplification, including my fragmente, however my intersted fragment is faint, I have made a temperature gradiente and dilution of cDNA, even with adding DMSO but I have not had succes.
If somebody knows how to improve the amplification I would be very grateful.
Thanks
Why dont you try to improve the primer design? You can try that primer becomes more specific. Make the BLAST to find which genes are the most similar to your gene of interest, make the multiple alignment and design primers in regions that you see are specific for your gene... My guess is that your current primer is not specific enough, check if your current primer bind to other genes. Good luck.
Maybe you need to make some changes in your pcr protocol, check the primers concentration, Tm, and MgCl2 concentration. In my experience this changes works very weell. Also is very important that you try with BLAST to find which genes are most similar too your gene target as Diana says. Good luck
options: 1. new primers from same of different coordinates 2. faint band excision from gel and reamplification Product sequencing in any case
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