We are working with pDUO2 plasmid and after transfecting it getting low expression of protein. I want to place A2UCOE sequence [HNRPA2B1 and CBX3] before the promoter for better expression, if any one can help me designing experiment to modify the plasmid will get a award of 200 USD.
thanks,
Girish
Hi Gannareddy
For any modification you want to do in your vector, I recommend you to use sewing PCR. It will help you to surgically remove or insert any sequence (fragment) you want by PCR. Here'is the strategy to follow. If you have any question, just ;let me know.
Get the IDT g block megaprimer (with 5' phosphate) with A2UCOE sequence and flanking sequence at 5' end homologous to plasmid sequence of choice (in your case just upstream of genes of interest). And also generate a Rp (typical ~20mer with 5'P) amplifying the rest of the back bone. Do the PCR. DpnI digestion to remove the template. Ligate and select. You should have the plasmid.
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