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I have ordered the primer.
Another question is about the extension time in secondary PCR.
It seems likely that vector but insert will be amplified in secondary PCR.
So should I estimate the extension time according to the size of original vector? Or insert+vector?
The extension time suggested in this web designer is quite different from my original thought.
Thank you.
Hi Hongrulin,
I've found that cleanup is actually detrimental; you generally lose some material in the process.
I use 1 µl of DpnI digested PCR mix in 20 µl of comp cells, so you can safely scale up to 5 µl in your 100 µl of cells.
I'll be very interested to hear from you if this project works out okay. I've never tried anything quite like it before.
Best of luck,
-Steve
Thanks for your kindly reply. I will update the progress.
Hi all,
Thanks this website for lots of technical assistance.
I'm managing to create a construct. Both insert and vector are about 5000bps. I know the efficiency may be low.
http://www.rf-cloning.org/rf_cloning_pr … 2e71bc32fb
Some basic question.
After DpnI digestion and inactivation I wonder if clean-up is needed before transformation?
How many DpnI-digested PCR reaction mix do you often use to transform to 100 µl competent cells?
Many thanks again!
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