Why is my insert missing from my plasmid? - (Oct/28/2013 )

Hi everyone,

I've just started at a biochem lab and I'm having real difficulty with protein expression!

I'm using the novagen LIC kit (vector = pET46 Ek/LIC) and have prepared my 600 bp insert according to the instructions that came with the kit:

1. Amplify target gene with LIC-compatible primers

2. gel purify insert

3. T4 polymerase treatment of purified insert

4. annealing insert with plasmid (I'm assuming that the plasmid is already linearised/T4-treated etc.)

5. transformation into competent E. coli cells (HMS174 DE3) on ampicillin plates

6. using cultures derived from a single colony for both midiprep (for sequencing), and IPTG-induction/protein expression

I checked some of the colonies I got from the transformation by doing a miniprep on each, followed by PCR with my insert-specific primers, then running the PCR products on 1% agarose gel. I got a band the correct size of my insert in the majority of the minipreps, so chose the one with the strongest band and used this plasmid DNA for a second round of transformations. This time I did a midiprep and sent it away for sequencing to verify the correct insertion/orientation of my insert before trying to express the protein.... it came back empty i.e. just the vector sequence, no insert!!!!!!!

Does anyone have any idea what I've done wrong? Is there any way I can check that my vector contains my insert FOR SURE, before I send away for sequencing again!? Don't think my boss will be happy paying for yet more incorrect sequencing data!!!!!! Please help, I'm really stuck!

Select colonies and screen by PCR using an insert- and vector-specific primer.

Hi,

Thanks for your suggestion jerryshelly1, I'll give that a try!

Was thinking of using a small amount (2ml) of each of my 100ml overnight cultures and doing a quick miniprep/pcr/gel to identify if any of them contain my insert before I do the midiprep for sequencing (cuz we have limited midiprep spin columns available)... does anyone know if it's ok to store an overnight culture in the fridge for a few hours while I'm waiting for the miniprep results? Will this affect downstream protein yields? Probably a dumb question, but I'm totally new to all these techniques!!!!!

We store them for up to 14days. After that, weird things can happen.

Has you lab ever considered switching to a crude phenol-chloroform extraction method to save money. This method can be done when trying to isolate ligation products and the midiprep columns can be saved when preping large quantities of the sample.

Edit - Storage will probably depend on your E. coli cell line. This is in reference to DH10beta. I haven't tested on additional cell lines.

I'll give it a go, a couple of hours can't hurt.... (hopefully)!!!

Thanks so much for your suggestions, money is a major issue in the lab at the moment; hence my fear of getting unsuccessful sequencing results back again!