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Filling with Klenow - (Jan/17/2008 )

Hi ,
I want to do a filling reaction with neb Klenow (large) polymerase for both plasmid and insert. In the NEB protocol they asked to add 33┬ÁM dNTPS each but nothing about the volume or about the total volume of the reaction mix. Can anybody help me to do the calculation for the dNTP concentration. I have a stock of 100mM dNTP mix (NEB). Should I do purification after this before proceeding to phosphatase as well as ligation reaction? Did anybody use Quick ligation mix (NEB)for blunt end ligation. I am planning to use it .
Thanks

-rohini-

Hi,

the way I calculate it is: concentration1 x Volume1=concentration2 x Volume2 (mol1=mol2). Concentration1 is 100mM, concentration2 is 33uM, Volume2 is the volume of your reaction and you want to know Volume1. If I do remember it right, you inactivate the Klenow fragment after the reaction and go on with the phosphatase. I would do purification before the ligation. Remember to use the phosphatase only for the vector. I haven't used the Quick ligation mix before, so I don't know. I think that an overnight ligation at 16 degree with the normal T4 DNA Ligase should be fine for routine ligations.

-zek-

The PEG 8000 in the quick ligase buffer, increases macromolecular crowding effect and aggregation, that enhances ligation..
it should work, if cost is not the factor...however if u already have the normal t4 dna ligase, u can try that first, and if that fails u can go for the quick ligase. the quick ligase buffer from neb comes as 2x and not like the regular t4 dna ligase 10x buffer..it means u need to take 10ul of 2x buffer in 20ul ligation reaction and u have to ensure sufficient concentration and ratio of both insert and vector and ligase in the rest of 10ul vol...

-Nagu-

Thanks zek and nagu.
I know the equation C1V1=C2V2 . Should I use the 33mM dNTP (each) as the final concentration? Most of the the time the protocols suggest to add xul of xnMdNTPS, thats why I got confused.

Now after reading your suggestion I think I will try T4 ligase first.

Thanks

-rohini-