filling in Pst I overhangs

Matt Thomas Thomas at molbio.uoregon.edu
Wed Oct 16 19:11:01 EST 1996


>I am trying to modify a gene by blunting the ends of Pst1 (gives 5'
>orverhang) and BamH1 (gives 3' overhang). We have tried to blunt the ends
>by using T4 polymerase to chew up the ends of Pst1 site and fill in the
>3' overhang of BamH1. However, we had no luck. Does anyone have any
>suggestions for us to try? Please specify the enzymes that you have used.
>You can post the answer on the net. Thank you.
>
>
Isaac.

I blunt Pst1 overhangs all the time and this is what works for me.

Digest with Pst1
phenol ext, etoh ppt with a 70% wash

resuspend the DNA
Add Boehringer Mannheim restriction buffer A to 1X
100µg/ml BSA
1-2 units of T4 DNA polymerase
250µM dNTP

10min at 37°C and then shift to 15°C for 20-30min
phenol and etoh ppt.

this gives me clean blunted ends all the time.  AS I understand it the
nuclease is very active at 37° and hardley active at 15° while the
polymerase activity is still going.  So the enzyme chews off the end
during the first 10 minutes and alittle bit farther and then when you
shift the temp things get filled in.

For filling in BamHI sites I just do the digest, don't even clean it 
up just add 1µl of klenow and some dNTP's and about 20 min at 21°C.  
Klenowworks in most restriciton buffers and unless you regenerate the 
site you can just fill in after the restriction without having to fill 
in.

Let me know if you need any more info.

______________________________________________________________________
Matthew Thomas,
Institute of Molecular Biology, University of Oregon
Thomas at molbio.uoregon.edu

Who has time for a snappy sig when you are having fun in graduate 
hell, I
mean graduate school!  :)
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