Hello,
So I am in the process of ligating RSVGN insert to pali2 vector.
I performed PCR for amplification of RSVGN insert. After purifying it, I performed double digestion with Ncol and Spe1 (same enzymes I used on Pali2 vactor), and proceeded with reaction clean up.
After all the purification process, the concentration dropped to about 3 ng/ul, which I have been told is enough to work with.
But for downstream experiments, I was thinking if I could successfully amplify the RSVGN insert (that i previously amplified, purified, digested, and purified again) via PCR, since I have the primers for this region anyway???
Hi,
I guess you can use the digested and purified RSVGN insert as a template for PCR as you have restriction sites included in the primers.
yes, you can use it as a template. However, I would say it is not worth of efforts and material because you can successfully clone it into your vector even if you have such low concentration (worked for me many times, although with different vectors).
Other possibility (if you wish to have higher concentration of your insert) is to sub-cloning the purified insert into vector with T (or U) overhangs like pGem or similar. If you used Taq polymerase for amplification which adds non-template As to the end of your product, you can use it for cloning straight forward. If you used Pfu (probably yes, since it is high fidelity) polymerase, you have to introduce it by incubation with Taq and dATP. The reason why to bother will all of this is that such cloning work very efficiently even with low amount of insert and you can amplify your insert in vector transfecting it to bacteria and isolating. But please note that for this you would have to repeat your PCR, since you already cleaved your product, or u can incubate it with Taq polymerase and mix of dNTPs.
I hope this answers your question, although you did not specified the downstreams experiments you wish to do. Good luck!
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