help with recircularizing a blunt-ended vector? - (Jan/15/2007 )
A lot of people here have trouble with recircularized vector contaminant; I have the opposite problem, as I want to recircularize a vector cut with sticky enzmes, and I can't.
I cut a vector with 2 sticky enzymes (EcoR1 and Cla1) and then inactivated the restriction enzymes by heating at 75C for 15 minutes; I filled the ends with T4 DNA pol + dNTPs directly in the restriction enzyme buffer; purified the vector on gel; and used 1/4 of it (maybe 200 ng) in a ligation reaction (20 ul, with T4 DNA ligase and the usual buffer and conditions).
It didn't work at all, as in: no colonies. What could have gone wrong? Should I phosphorylate the ends at some points? Should I use more DNA, or less? Thanks for any tip.
ends are phosphorylated as they're produced by restriction.
Try to do klenow in appropriate bufer.
I would gel purify after digesting with both enzymes, blunt end it ( i used klenow) , inactivate it and use around 10-20ng for ligation reaction.
My lab has never gotten good results using T4, we always use Klenow. This is how I recircularized my vector recently:
A. digest vector with AatII and XhoI
B. blunt with Klenow for 15 min at 25c
C. isolated cut vector on gel (0.8% agarose)---->electroeluted linear vector
D. put 1ul of purified vector through ligation rxn using NEB Quick Ligation kit.----> got >100 colonies
I cut a vector with 2 sticky enzymes (EcoR1 and Cla1) and then inactivated the restriction enzymes by heating at 75C for 15 minutes; I filled the ends with T4 DNA pol + dNTPs directly in the restriction enzyme buffer; purified the vector on gel; and used 1/4 of it (maybe 200 ng) in a ligation reaction (20 ul, with T4 DNA ligase and the usual buffer and conditions).
It didn't work at all, as in: no colonies. What could have gone wrong? Should I phosphorylate the ends at some points? Should I use more DNA, or less? Thanks for any tip.
u need to phosphorylate the ends in order to self ligate the vector. T4 DNA poly treatment leaves ends unphosphorylated. u have to treat your vector with T4 Kinase before ligation.
I cut a vector with 2 sticky enzymes (EcoR1 and Cla1) and then inactivated the restriction enzymes by heating at 75C for 15 minutes; I filled the ends with T4 DNA pol + dNTPs directly in the restriction enzyme buffer; purified the vector on gel; and used 1/4 of it (maybe 200 ng) in a ligation reaction (20 ul, with T4 DNA ligase and the usual buffer and conditions).
It didn't work at all, as in: no colonies. What could have gone wrong? Should I phosphorylate the ends at some points? Should I use more DNA, or less? Thanks for any tip.
u need to phosphorylate the ends in order to self ligate the vector. T4 DNA poly treatment leaves ends unphosphorylated. u have to treat your vector with T4 Kinase before ligation.
This is not true. T4 DNA poly does not leave ends unphosphorylated. I think you are confusing this with the primers that come from the manufacturer unphosphorylated. Therefore, PCR products generated with such primers have to be phosphorylated with PNK prior to ligation. I digested a vector with SalI and then filled the ends with T4 DNA Poly and was able to recircularise with no problem. Here's what I did:
1. Digest vector with enzyme
2. Precipitate DNA with ethanol and wash pellet with 70 % ethanol once.
3. Resuspend pellet in water and add the following;
- pfu buffer
- MgSO4
- dntp's
- 3 units of PFU
4. Place tube at 74 degrees for 15 minutes and purify
5. Circularise by ligation
Be aware that the buffer and concentrations of MgSO4 and dNTP's can affect your reaction. Go to this reference:
Yang, S.. et al. 2005. A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase. : Biotechnol Appl Biochem. 42(Pt 3):223-6
Basically, the highest filling-in efficiency for pfu is in a buffer (pH 8.5) containing 3 mM MgS04 and 300 uM dNTP at 74 degrees. According to this manuscript, this is 2 times more efficient that Klenow.
This is not true. T4 DNA poly does not leave ends unphosphorylated. I think you are confusing this with the primers that come from the manufacturer unphosphorylated. Therefore, PCR products generated with such primers have to be phosphorylated with PNK prior to ligation. I digested a vector with SalI and then filled the ends with T4 DNA Poly and was able to recircularise with no problem. Here's what I did:
1. Digest vector with enzyme
2. Precipitate DNA with ethanol and wash pellet with 70 % ethanol once.
3. Resuspend pellet in water and add the following;
- pfu buffer
- MgSO4
- dntp's
- 3 units of PFU
4. Place tube at 74 degrees for 15 minutes and purify
5. Circularise by ligation
Be aware that the buffer and concentrations of MgSO4 and dNTP's can affect your reaction. Go to this reference:
Yang, S.. et al. 2005. A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase. : Biotechnol Appl Biochem. 42(Pt 3):223-6
Basically, the highest filling-in efficiency for pfu is in a buffer (pH 8.5) containing 3 mM MgS04 and 300 uM dNTP at 74 degrees. According to this manuscript, this is 2 times more efficient that Klenow.
[/quote]
upps! you are right 
I am sorry if I mislead anyone of you. 