Impossibly small ligation products
Posted 07 September 2004 - 11:42 AM
I don't believe I am contaminating with another plasmid.
I thought it might be a cryptic rearrangement of the DNA, but someone else in our lab just got similar results (ended up with more like 2.0KB plasmids) using a different vector (5KB) and insert (2KB).
Any ideas anybody?
Posted 13 September 2004 - 05:28 AM
when you digest by Klenow, use VERY little amount....
Posted 13 September 2004 - 07:01 AM
cut gel after digestion?
does the band positions are correct?
Posted 13 September 2004 - 07:26 AM
I extracted plasmid using a fairly standard alkaline lysis protocol with isopropanol precipitation. I ran some on a spec and got a good yield with low 230 and 280 readings.
I purified from the gel (0.8% Seakem GTG, 5v/cm, 1hr) using an Eppendorf Perfectprep kit (but I have also tried the Zymoclean kit from Zymogen and a homemade column). I ran products of this and got good bands (size and intensity).
I used the Klenow from NEB (Cat # 0210). I diluted to get the recommended concentrations of everything (diluted enzyme in 1x buffer), and ran the reaction for 15mins as specified in the instructions. I skipped the heat inactivation because I did a phenol:chloroform cleanup and precipitated again to be sure I didn't have any salt or enzyme carryover. I ran some of this on a gel and got a pretty good yield.
The other cloning that has given strange results in the lab did use Klenow.
I am currently religating the vector without the old insert, so that at least I won't need the gel to clean it up after digestion in the future. I might even be able to find a blunt cutter to use on it if this works, which would be one less thing to go wrong...
So you think it's the Klenow? Do you use less than the manufacturer's say?