You can check the plasmid you isolated on an agarose gel. Do some restrictions (single cut, double cut) and compare the fragment sizes you get to a map of the plasmid. If these match you can be pretty sure that you are working with the right plasmid. You said that you can only get approx. 1000 bp by sequencing. A single sequencing run will give you around 1000 bp of information if everything goes well. How many sequencing reactions did you do to map your plasmid? Your transformation protocol looks fine to me. There can be many reasons why a ligation fails. Why do you think that the vector is the problem?

Regards, Christian

Hi, thanks for the reply.

I'll definitely try the restriction digest. I've done a few sequencing reactions but at different points on the plasmid, so from T7 and the T7 term in the hope they would overlap but my previous transformation results were poor and I was getting less than 300bp sequencing with a lot of mismatches.

As to why I think the vector is the problem; I've been testing everything during my cloning on agarose gels/nanodrop and the insert always gives a strong band with good concentrations, whereas I get poor results from the vector. I checked the restriction enzymes and they weren't the problem. So I've been trying different ligation ratios and temperatures but to no avail. I understand that ligation can be tricky but I have troubleshooted everything else and the only thing left is the vector.

Thanks,

Holly

I see. The poor sequencing results indicate that there might be a contamination or impurity in your vector aliquot. This could also make your cloning efforts almost impossible. The 260/280 and 260/230 ratios were poor in the Nanodrop measurements? What are the values?

Cheers, Christian

For some previous attempts I've had 260/280 of 2.15 and a 260/230 of 2.37. Not optimal but ok.

But some have been 0.88 (260/280) and 1.25 (260/230) or even 2.27 (260/280) and 0.36 (260/230).

I put some of these down to contamination during miniprep.

My most recent results are probably the best with 1.92 for the 260/280 but a low 260/230 at 1.60.

Thanks.

I'd say that 1.60 for 260/230 is actually quite good. When you have really bad values for 260/230 you might run a dialysis for your plasmid. It is quite astonishing how much that can improve your transformation efficiency.

Regards, Christian