Ligation over a six day period - WOuld this have damaged the DNA? (May/05/2006 )
I have been doing ligations and transformations for two months now with little success.
I am trying to ligate a 800bp gene into pQE30.
Recently i set up a ligation / transformation and after it not working, I immediately set up another with higher insert:vector ratio. However, because I stuffed up a calculation, the ratio was only slightly higher than the first go.
I incubated the ligation at 4 degrees, but the next day decided it was not worthwhile to plate out the transformation and that I would wait until I was doing another one before going ahead with it.
It was six days until this happened, and my ligation sat at 4 degrees for this period of time. Yesterday I plated it out and got about 80 colonies, and have performed PCR to confirm presence of a fragment of the right length. I have found one that is the right length amongst several that contained self ligated vector.
Should I be concerned at all about what ligating for a week might have done?
I am planning to do a digest next week to confirm presence and thought I would perform another PCR using gene specific primers.
Any comments would be greatly appreciated
No. I always save my ligations, and have on several occasions gone back and re-done transformations using the same ligation after it had been held for weeks at 4°C. Never had a problem...
Should not be a problem at all. I have repeated transformations from the same ligation kept in RT for more than a week. And it does work.
What about leaving your ligations at -20 degrees for a few days...is that ok?
Like the guys have done, I have also stored ligation mixes at -20 degrees, thawed them out and re-done transformations and its been fine. Take a deep breath and relax, your experiments will be just fine