Problem in dephosphorylation - (Mar/29/2007 )
Hellow all I'm new to this forum, i joined this forum thinking it could give me a solution to the problem i'm facing right now.
I'm trying to dephosphorylate a newly constructed and linearized plasmid 7451bp, this plasmid is linearized using enzyme AatII. my problem is this plasmid keeps on religating eventhough i tried all means to dephosphorylate it completely. I used Antarctic alkaline phosphatase (Biolabs), SAP(Roche) and CIAP (promega). In all these treatments the control plasmid ligation is giving me lots of colonies i don't get it! 
Could anyone please help me out! 
Thanks indeed
Eminov
Use primers to amplify your cloning vector. The primers can contain whatever you want for restriction sites. Isolate the PCR product, cut with the enzymes of choice, and enjoy zero background (well, a little perhaps from the template). You can add DpnI if you want to get rid of the template.
Thank you indeed the idea of amplifying the cloning vector seems so brilliant, the thing that i don't get clearly is the use of enzyme DpnI (Methylation sensitive?). what does this do exactly? if you don't mind explaining me more!
Thank you again i'll go for amplifying my vector!!! 
well, phage434's idea is so smart, but i don't think it will work so well , because your vector is much too big for you,if your trouble vector is less then 5 kb ,it will be perfect.
My suggestion is that you can reduce the amount of vectors.I think set the ratio 100-200ng / 20U enzyme, then dephosphorylate will clear your control.
Because the plasmids in E.coli are always methyliated( very few excepts ), so DpnI can digest it and left intact pcr products.
goodluck
One possible explanation: when you get extra bands that migrate faster than supercoiled DNA on an agarose gel and are resistant to cleavage by restriction endonucleases, the DNA was irreversibly denatured. This has been observed when plasmids were isolated by the alkaline Iysis method. To minimize denaturation, use 0.1 N NaOH instead of 0.2 N NaOH and decrease the time of NaOH exposure. Irreversibly denatured plasmid is still capable of transforming E. coli.
If the alkaline lysis step is allowed to proceed for longer times or at higher temperatures, irreversibly denatured DNA will result. (J. Vinograd and J. Lebowitz 1966. Physical and topological properties of circular DNA. J. Gen. Physiol. 49: 103.) This material travels just below cccDNA on an agarose gel, stains poorly with ethidium bromide, and can comprise approximately 3% of the total DNA. This material is not cut by restriction enzymes, but it can transform bacteria. Thus it is a source of background in transformation that cannot be eliminated by extended restriction digestion or dephosphorylation. Treatment with T5 exonuclease, which possesses single-stranded endonuclease activity and 5’-exonuclease activity reduces the transformation efficiency of denatured DNA by two orders of magnitude. ( J. R. Sayers as cited in Methods and Reagents 1996 TIBS 21 441-442.) This denatured DNA is thought to be double-stranded, cyclic, coiled DNA composed of two intertwined, but permanently denatured, single-strands of DNA.
Of course, your linearized plasmid may simply be contaminated with uncut DNA. Do you run a transformation control consisting of vector only without ligase? And I have seen E. coli capable of generating hundreds of colonies from HinD III-cut vector (no ligase) unless the linear DNA was dephosphorylated.
SAP and Antarctic phosphatase work in most restriction buffers, so I routinely dephosphorylate during the restriction cut (if it is performed at 37C). This saves time and eliminates intervening cleanup steps that might result in loss of some of the DNA.
I'm trying to dephosphorylate a newly constructed and linearized plasmid 7451bp, this plasmid is linearized using enzyme AatII. my problem is this plasmid keeps on religating eventhough i tried all means to dephosphorylate it completely. I used Antarctic alkaline phosphatase (Biolabs), SAP(Roche) and CIAP (promega). In all these treatments the control plasmid ligation is giving me lots of colonies i don't get it!
Could anyone please help me out!
Thanks indeed
Eminov
Hi there. I experienced similar problem before. Probably you may want to try with what I have done previously. I dephosphorylated my vector with CIAP (Promega) and I followed the protocol for blunt end dephosphorylation. Compare to the sticky end protocol, I get no colony in my vector control plate.
Good luck.
DpnI cuts at GATC sites in which the A's are methylated. This only happens when the DNA is present in dam+ (most) strains of E. coli. Notably, it does not happen in PCR reactions. So the plasmid used as a template likely has methylated GATC sites which are cut by DpnI, while the PCR product is guaranteed not to. You cut the template, and leave the PCR product. The Stratagene mutation kits use this method to eliminate template in the mutation reactions.
thank you Everyone! after gathering these informations i decided for one last time to dephosphorylate my plasmid using AP but this time i incubated my linearied plasmid 1hr at 37°C with 5U Ap, and i proceeded the same procedure three times. at the end of the dephosphorylation process I quantified my plasmid which i found out to be 10ng/µl. then i did 5:1 ligation reaction with my insert 2.2Kb (5ng/µl). i.e my insert size is less than half of the vector size which is 7.5Kbp.
When i migrate on gel 2µl of my ligation product i found out 6 different size concatemers.
transformation of vector only dephosphorylated gave zero colonies this time, transformation of insert vector ligation however did not produce any colonies after 16hrs of incubation at 37°C. 
Though i succeded in the dephosphorylation i dont understand now what had happened with the ligation or transformation steps! looking forward for a solution!
Eminov