Dear cloning experts,
I run out of ideas so I think it's time to ask help to the community.
I need to clone two chicken related transcription factors (TFs) into an rcas vector (avian retrovirus). I have first cloned these two TFs into an intermediate vector to add a tag on the 3'-end. I have checked my both constructs by sequencing, everything is fine.
I digest my constructs (1 kb) and my empty rcas vector (11.5 kb) by using 2 different restriction enzymes overnight, followed by a dephosphorylation for the rcas vector. I run my digestion products on a gel, cut the bands of interest and perform a gel extraction by using columns. Then, I ligate at 16°C overnight and transform competent TOP10 cells by heat shock. On the next day, I get from 5 to 20 colonies and I analyze them by PCR by using primers on the vector surrounding the insert location, in order to make difference between vector-only and vector+insert clones.
For one TF, I got my final construct after couple of tries but for the second TF, it keeps failing. I have tried many different conditions for the ligation (different ratios insert:vector [from 1:1 to 10:1], always using fresh aliquots of ligase buffer and ATP, add PEG or not) and for the transformation (30 min on ice with the ligation product, from 30 sec to 2 min at 42°C, 10 min on ice, 1h at 37°C in SOC or LB). I have tried different competent cells as well such as SURE2 and XL10-Gold. Test my restriction enzymes which work fine.
For this second TF, I get in general a few colonies and I don't amplify any product after the PCR with the same primer set. I use as positive control one of the positive colonies that I got for the first TF. I always get a band for this control, so it doesn't seem that the PCR fails. I have tried twice to do a few mini-preps from random colonies and perform a digestion test. I got an unique band for which the size doesn't correspond to anything that I should get...
The only reason that I have in mind would be that my cells catch the plasmid, get the resistance to the ampicillin and then release the plasmid. I don't see any other explanations (but I'm not a cloning specialist). I think that it's important to notify that I use competent-cell aliquots and LB+Amp plates from the same batches than my other coworkers, who don't have any trouble like mine for their cloning.
If anyone has any idea of what is going wrong, what could I try or test, or whatever, please share your ideas! I start losing my mind!!
I will be very grateful for any help. Cheers,