subcloning of PCR product, please help! - subcloning problems (Jun/07/2005 )
Aint it a bitch when a simple subcloning scheme goes sour?
Finally, I have some good news!! I'm so happy to tell you that... my cloning finally worked, after two-and-a-half months of "CLONING HELL" and after i started wondering if the protein is toxic to e.coli (so the recombinants would be selected against).
Here is what I learned. (Again, this is in context of cohesive end cloning of a PCR product (with restr. sites located 3-4 bp from the ends))
The main thing is that LIGATION is the trick. I did extensive controlled comparisons of different reaction conditions and different ligases, and there are TWO main conclusions...
1) NEB CONCENTRATED LIGASE is (by far) the best. Don't hesitate to do overnight ligations (they work MUCH better), preferably at 15oC.
I heat-deactivate (10 min at 65) after 16-24 h, but I'm not sure how critical that is. I do the reaction in 10 ul total with 0.5ul of ligase and then use 1.5ul for transformation.
2) INSERT at HIGHER concentrations than usually recommended - insert-to-vector ratio of something like 10:1 and higher makes a difference.
You don't need more than 50 ng of vector per ligation.
(You can use this great site:
to instantly calculate the ratios based on mass and size)
P.S. concentrated DNA ligase NEB catalog # :
M0202T (20,000 units 2,000,000 units/ml $63.00)
Congratulations, we have all experienced the cloning hell. For something that should be so simple cloning can sure generate grey hairs. Also thanks for the tips and the link to the great site.