David Smith

Moredun · Department of Disease Control

I'm baffled.

On numerous occasions now, I have failed to gain cells on an LB amp plate from transformed TOP10 E. coli cells.

I have attempted various strategies to figure out what the problem is:

My insert gene is small (~200bp) and I normally get low yield, thus I have tried scaling up by restriction digest and I have attempted a PCR of my insert prior to restriction digest to ensure there is plenty of DNA. This works, I get a much brighter band showing up on an agarose gel, at the correct size for my insert too.

I have also ran the the products of single digests, double digest and undigested insert on a gel to show that both enzymes are working effectively - and they appear to be, whereby in the double digest I get the product I am looking for to purify and ligate. I have also done this for the Ppinkalpha-HC vector that I am ligating into, to show that this is also cutting with the enzymes.

I have tried eluting the products from the gel purification into sigma water and elution buffer.

I have tried various ligation ratios.

I carry out my ligations at 4C overnight, but I've also tried 15 min room temp.

Most puzzling of all is that I have attempted to carry out this work alongside a colleague who is doing the same work with another gene (theirs is ~400bp). I did this to ensure I wasn't making any silly mistake or anything. Thus we followed the exact same protocol (even use the same RD enzymes), at exactly the same time, alongside each other.

Low and behold, they gained some colonies on their insert+vector plate, whereas I gained none. We both also ran a control with just the vector in the ligation mix (no insert gene) and transformed this. I gained now colonies, whereas my colleague gained a lot of colonies, suggesting the vector had self ligated and got into the cells, providing ampicillin resistance and allowing colonies to form on the LB amp plates. Although colonies on her negative plate are not desirable, this showed that the ligation is working. My negative ligation sample is the exact same mix, yet I gained no colonies. Given that my colleague did gain colonies, there's liklihood that the same self-ligation of the vector occurred in my negative control, which makes me think my ligations should be working. Thus perhaps it is the transformation that is not working?

But why, given that I carried this out alongside a colleague, following the exact same protocol.

Cells were not killed by the sterilised spreader, it was allowed to cool. Cells are new. The cells are bought in from life technologies already chemically competent.

Some other details:

I heat-shock cells for 30 seconds at 42C.

Ligation mix material works (as shown by my colleagues positive results).

The gene I am working on is very similar to two other genes I have previously been successful with, the only difference being 3 amino acids in the expressed protein sequence. That's the other thing that puzzles me, I did not run into this problem with two other very similar insert genes, which had the same restriction digest sites, etc.

If anyone has any suggestions, I'd really appreciate any help and advice!