Unable to obtain colonies from PCR-insert + vector ligation - (Jun/14/2006 )
I am using a pET-19b (5.5kb) and a 1.4kb insert, which I attain using overlap extension PCR.
I digest my vector using BamHI and NdeI (together), then dephosphorylate (in same tube) using CIAP. I purify the vector from agarose gel using Amersham Biosciences purification kit.
I also purify the insert after the last PCR reaction (PCR 3), then digest it with the same REs and purify from gel again.
My final concentrations are: 16ng/ul vector, 64ng/ul insert.
I've tried molar ratios of 1:3 and 1:6 (vector:insert).
I use the Quick Ligase kit from NE Biolabs, incubating my ligation mix (20ul total) for 5 minutes at room temperature, then use 2ul of the ligation mix with 50ul competent cells for electroporation-transformation.
The competent cells are alright, as I've positive-checked them, and they've been used successfully by others.
Any suggestions or remarks will be greatly appreciated.
Last time, which is more than a year ago, I used the quick ligation kit (NEB), they suggested to heat shock cells and not electroporate. So check if this has changed.
From the NEB website:
Electroporation: Electroporation can increase transformation efficiency by several logs. Before using the products of a Quick Ligation reaction for electrotransformation, it is necessary to reduce the PEG concentration. We recommend a spin column purification.
So: try heath shock transformation, or purify your ligation, by spin column or precipitation.
Thanks for the tips.
I tried getting rid of the PEG in the quick ligation mix using spin columns, and then transforming using the purified ligation mix (w/o PEG).
Still no results.
Any other suggestions? I'm still very stuck here...
If u could borrow a few chemical competent cells for someone, try heat shocking the cells.
Thanks. I tried heat shock, didn't work. Also tried ligation with a regular T4 DNA ligase (not the quick ligation kit), didn't work.
I think maybe the problem is in the ligation?
If none of the ligations worked, I guess the problem might be the overdigestion of the vector or insert. Could u tell more abt ur digest conditions.