klenow/T4 question - Making one end blunt (Sep/17/2007 )
I just realized I am out of practice with my cloning techniques. I have a fragment that I need to move from a vector by Eco R1. The way the original vector was cloned such that there is an R1 site on either end of my gene. I was hoping to keep the cohesive end on the 5' end and blunt the cohesive end on the 3' end. See below
After R1 cut I have:
5' AATTCGACATG...ACG
3' _____CTGTAC...TGCTTAA
I want to make it into:
5' AATTCGACATG...ACGAATT
3' _____CTGTAC...TGCTTAA
or
5' AATTCGACATG...ACG
3' _____CTGTAC...TGC
But I don't want to make:
5' AATTCGACATG...ACGAATT
3' TTAAGCTGTAC...TGCTTAA
The vector I want to clone into works well when cut with R1 and SMAI which are cohesive and blunt respectively, therefore, I want to keep the cohesive R1 from the insert and blunt the end so that when I clone it I won't have to worry about orientation of the insert.
I have Klenow(DNA polymerase I large fragment), but I assume this will create both ends blunted.
I also have T4 and my protocol says it removes 3' overhangs and fills in 5' overhangs, but I don't think that will get me what I need.
As long as the "top" DNA strand has a 5' cohesive R1 end and a blunt 3' end it will work.
It seems I've done this before, but it has been 5+ years and I can't remember the trick. Can someone help? Or should I just blunt both ends and play the orientation screening game?
Thanks!
Can you use a different enzyme to make the cut at the 3' end of the insert? You need at least 2 different enzymes to selective blunt one end of the molecule. And in this situation the unique enzyme must be behind the EcoRI site to avoid it being cut off.
If this is possible with the vector's SmaI site, can you not play the orientation game with the EcoRI. Blunt end cloning is more difficult then having to orientate your insert.
I don't think I have a chance to use a different enzyme for the 3' cut. The person who initially cloned the insert into our vector put R1 sites very near the start and stop codons which pretty much takes any other MCS enzmes out of play. When I first started this project I saw the R1 site before the start codon and a RV site after about 50 bases after the stop codon, but didn't notice they added a R1 site about 10 bases after the stop codon, so that made my R1/RV approach useless.
Maybe I'll have to get creative with this one
Well there is always PCR. ie PCRing your insert with primers that add desirable unique restriction sites
But an EcoRI to EcoRI; insert into vector ligation certainly looks doable.
Looking into this further I found that I could move my sites a bit and use Bam/RV sites in the insert and BglII/Sma sites in the recipient vector for a compatible cohesive end 5' and a blunt end 3' ligation.