ds-oligos cloning problems - 90bp oligo and 12kb plasmid (Sep/24/2009 )
Hi, I am trying to clone a 90bp oligo into a 12kb plasmid. My plasmid has a single cut by SpeI, and my oligos have complimentary overhangs.
For the vector, I digest and dephosphorylate and then do enzymatic cleanup using Qiagen kit. For the oligos, I anneal with annealing buffer (tris, edta, nacl), boil for 5 min and let it cool overnight, then I clean up using Qiagen kit and phosphorylate with PNK.
For ligation I add digested dephosphorylated vector and phosphorylated ds oligos with Roche Rapid Ligation kit, incubate 15 minutes at room temperature.
For transformation I electroporate using default settings for E Coli, 1mm cuvette (time constant ~5ms, V~1.8kV), then I let it incubate for 1 hour at 37 degC 250 rpm. I use 20ul of cells and 2ul of ligation mix, then add 1ml SOC after electroporation. I have tested out the competency of my cells and they are 5x10^8. For the positive control using uncut plasmids, I get a lot of single colonies. For the negative control with no insert and only the dephosphorylated vector, I get no colonies. I usually spin down my transformation mix and resuspend in 100ul to plate on LB Amp100 plates.
I have tried different molar ratios of vector:insert 1:1, 1:3, 1:5, 1:10 and none of them have worked. I do not get any colonies at all. Does anyone have any ideas on what else I can troubleshoot?
Do you know that your ligase is working well, and that the insert is correctly phosphorylated? Try a quick self-ligation with the inserts, and also do the same with DNA ladder. Even 15 minutes at RT will be enough, then run on a gel. If you don't have a ladder of insert, and a shift in the DNA marker ladder, your ligase is sick/off/dead. If the DNA marker works, but the insert doesn't, then your PNK isn't working well, or your ATP is off.
I would also try a longer ligation reaction in the cold.
ds-Oligo cloning is not that hard and should be very straightforward, but 90 bp is very long for oligo cloning so make sure you have a reliable oligo synthesis provider -- the oligos you get should be full length!
Important: After annealing and ligation, you should run argrose gels to check whether the annealing and ligation are successful. By running the annealing products alongside with ss-oligo, you will also know if the oligos are high quality (full length).
You can also try ligation at 4C overnight.