Hi, I have been doing PCR to the cDNA extracted from cell, but I need one gene to be amplified from those cDNA library, how can I purify my target amplicon from all of those dscDNAs?
I have been extracting the target amplicon from the agarose gel directly according to it's size, but concentration is very low in this case. Can anyone suggest a better method to purify my target DNA amplicon?
Try doing a NESTED PCR (i.e. add a secondary PCR with a nested or internal primer to increase both quantity and specificity of the amplicon to be cloned).
Try increasing the Mg Concentration of your reaction to aid better primer binding.
It is just for my clarity..how you prepared cDNA from cell..like first you isolate RNA and then you prepare cDNA. If your target cell is prokaryotic then you need either gene specific primers or random hexamers to prepare cDNA from mRNA.If your target Cell is Eukaryotic then you can use oligo dT method.
Now when your cDNA is prepared just go for specific qPCR (by using specifically designed primers) and then purify your PCR product by any good PCR purification kit.
Hi Hediche, If you know the sequence of your protein of interest, you can whistle up a couple of primers... best make it at least 4.. two for the 5' end and 2 for the 3' end. then purify using Qiagen PCR cleanup spin columns.
regards
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