Best way to inactivate Klenow fragment? - (Jul/26/2007 )
Hi.
I am planning to cut a vector with SalI, blunt the cut site with Klenow fragment, then cut with EcoRI to generate a sticky end (result - one blunt, one EcoRI cohesive terminus). I want to make sure that Klenow is inactivated/removed from the mixture before the second cut and does not work on EcoRI ends. What is the best way to do it?
1) Purify the vector with Qiagen columns?
2) Heat inactivate it? How reliable is heat inactivation (NEB says 20 min at 75C)? I would prefer heat inactivation, since I am having problems with purification colums, but want to make sure it is 100% efficient.
Thank you for your thoughts.
may you tell us what are your column problems???
i would rather column purify than taking the risk of a residual klenow activity in my preparation....
moreover 75°/20' isn't not that friendly for DNA ? what do you think others?
column tips : if you tend to loose DNA or bad eluate, make two elutions and then reduce volume at spped vac.
Second, make sure to wait enough before elution after the column wash step. Protocols that mention 2 spinning are sufficent to dry columns are making experimenter to get impourities or solvents in elution fraction (check OD230 for that.)
We have used klenow and heat inactivated it. It has worked. Ofcourse, it can be tricky.
If you have a lot of DNA, better to purify it by columns. You could have 1-2 bad columns but generally Qiagen columns are good.
fred_33: the problem with my column purifications is that enzymes (restrictases, ligase) do not work on DNA once it has been run through a column. People have suggested max speed dry spin to remove all residual ethanol before elution, but it does not help. I don't know what causes the problem and how to solve it.
Thanks for all suggestions regarding Klenow inactivation, I will try.
How about phenol/chloroform extraction and ethanol precipitation? This can remove both Klenow and dNTPs from your DNA sample, and the EcoR I cohisive ends could not be blunted (filled) by Klenow without dNTPs.
By the way, heat inactivation is also a choice.