Cloning into pCS2+HA - suggestions needed please

[genotype]

Hi everyone,

I am new here and hoping someone can give me advice on cloning issues I am having. I am currently generating three clones, which are chimeric genes. Basically, I am using PCR to create fragments of the domains of the gene from two different species and then doing a second PCR to bring the fragments together (each fragment has ends that overlap the adjoining fragment). I am not having any trouble with this and have been able to clone all three of my constructs into pGEM-T without too much difficulty. I am now attempting to subclone these into pCS2+HA (a pCS2+ vector with HA tags added) and have successfully done this for 2 out of 3 of my constructs. However, I am getting very low efficiency, and so far have been unable to get the third construct working. I will outline my protocol (starting from the pGEM-T constructs) below. Any suggestions would be much appreciated!

1) Use PCR to generate the gene with restriction sites (XbaI and XhoI). The primers I am using have 6 additional nucleotides prior to the restriction site.
2) Cut fragment with XbaI and XhoI (at least for four hours, if not overnight). At the same time, cut pCS2+HA vector (I have done a control where I cut with each enzyme individually to make sure they are cutting).
3) Phosphatase cut vector. (I have used phosphatased vector that someone else in lab used successfully for pCS2+HA cloning as well - this also did not work).
4) Gel extract vector and fragment.
5) Do ligation - I have varied the insert:vector molar ratios from 10:1 all the way to 1:10. I use 1 microliter of ligase in a 10 microliter reaction.
6) Leave ligation mix at 16 degrees overnight (I have also tried room temp for 2 hours and 4 degrees overnight).
7) Transform into DH5alpha competent cells. Leave overnight.
8) Check colonies using colony PCR (I am getting less than 1/50 efficiency!). I use a primer specific to the insert and one for the vector to avoid picking up background.
9) Check + colonies with restriction digest and, if it looks good, sequencing.


I noticed that the number of neg. control colonies (self-ilgated vector) is not much different than that on the other plates. Also, I plated cells only to make sure the ampicillin in the plates is good -- nothing grew.

The construct I am still trying to get does seem to interfere with plasmid growth as I get very low concentrations upon miniprepping the pGEM-T version (40-50 nanograms/microliter as opposed to 200+ for other constructs).

Please let me know if you have any suggestions. Thanks so much for your time!

Genotype