help! t4 kinase reaction - Does SDS inactivates the enzyme? (Aug/12/2005 )

Hi everyone,
i performed a T4 polynucleotide kinase reaction, unsing Promegas reagents. I performed the whole thing "by the book", but in the inactivation step I used SDS instead of EDTA as suggested. I noticed afted phenol:chlorophorm and EtOH pp that no pellet was visible. Do you think this would interfere with my DNA recovery?This fragment will be used in a ligation reaction further.

Please!!! any comments on this will help!

Dear brother,

I always stop the reaction by vortexing after phosphorilation without add any reagents, and used it directly without purification. Could you please give me information the purpose of your reaction, your reaction mixture and reaction protocole cronologically? The amount of ATP that you used has correlation with the time of incubation during reaction.

warm regard
Ali

Hi Ali,
I was trying to phosphorilate a PCR blunt fragment obtained with Pfx amplification, in order to clone it into a appropiate dephosphorilated vector.

For the T4 kinase reaction I used:
- 90ng 500bp DNA
- 10x kinase buffer 4uL
- 0.1mM ATP 2uL
- T4 kinase 15U
- ddH2O to 40uL
Incubation at 37°C for 30 min.

Anything you can ad to this will help!
myclone

You have a pretty low final concentration of DNA. You might want to add a carrier like glycogen before the EtOH precipitation if you are worried about losing your DNA.

Daniel

DNA sequencing reagents

hi
SDS is not the best way to reciver DNA by i thionk it will interfers with T4PNK.
After reaction, i directly precipiate the DNA.
But 90ng of DNA is very few...
I'm not sure it would be visible even with a good precipitation?!...