RF-Cloning.org Forum

hongrulin

Member

Registered: 2015-02-04

Posts: 3

Procedure after DpnI digestion

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!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Procedure after DpnI digestion

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

hongrulin

Member

Registered: 2015-02-04

Posts: 3

Re: Procedure after DpnI digestion

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.

hongrulin

Member

Registered: 2015-02-04

Posts: 3

Re: Procedure after DpnI digestion

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.

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Procedure after DpnI digestion

Sorry for the slow reply hongrulin, I didn't get my usual email alert on this for some reason.
Anyway, for the 2° PCR reaction, you only need to extend along the destination vector. The insert was synthesized when you made the megaprimer in the 1° PCR reaction, and will join up with its complement later.
Regardless, go ahead and increase the extension time by ~30%. I've been playing with this recently, and a little more time at 74°C seems to help.
-Steve