Blunting with klenow fragment - (Mar/09/2006 )
Below is my cloning problem. I hope someone can please help me!!
Vector to be cut with Sac II, blunt it with Klenow fragment, ligate with a Tetracycline PCR product and transform into Pseudomonas aeruginosa and plate on Tc 50ug/ml. I have done the above but there were no colonies at all.
My questions are:
-Can Klenow fragment be used or T4 DNA polymerase would be a better choice?
-Do I need to gel purify after each step? (SacII and Klenow fragment are from Fermentas and T4 DNA ligase is from NEB)
-Tc concentration too high? (One of the postdoc had adviced me to use Tc 50ug/ml)
I have previously used the same parameters for the electroporation and there were no problems. I had to do this blunt end cloning as I do not have the sequence to the pcr product. It was given by someone.
I would cut with SacII and then use Klenow fragment. I don't know if the buffers are the same, but otherwise I would for example do the restriction in a total volume of 10µl and then add to a total volume of 50/100 µl for the klenow. After this, I would purify it from a gel, also to check the quality of the DNA after the klenow threatment.
I don't know the the Tc concentration for pseudomonas. Try finding it on the internet or ask someone, or maybe it is in a manual.
I'm agree with aspergillie togeather with the following..
Klenow treatment could remove 3` overhanging ends, but for this purpose T4 is more popular because of the reaction speed, however T4 polimerase is realy agressive and could remove several nucleotides from the end of the DNA fragment even when it is blunt ended (that is why i do'nt like it). Interestingly I could use the same Klenow treatment to blunt DNA fragments wiht 3` and 5` overhanging ends simultaneously wihtout any problem. Simply I add Klenow fragment and dNTPS to the digestion mixture, and I let it work for half an hour. After heat inactivation and fragmentisolation. (normalwise it is unnecessary to make them togeather)
The only thing you should keep in mind that the concentration of glycerol in the reaction mixture shuld'nt reach more than 10%!! So if you use 30ul of reaction volume the maximum volume of the enzimes shuld be below 3ul, if you use fermentas products, wiht the high glycerol storage buffers.