Filling with Klenow - (Jan/17/2008 )
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 33µM 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 .
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.
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...
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.