Tips on improving ligation+transformation from gel extraction - Let's contribute! (Jan/01/2008 )
After gel extraction, normally subsequent reaction wouldn't be very pleasant. Here i would to share my experience and asking everyone in the community to contribute so that we can help making the lab a better place to be in.
RE digest-->Gel extraction --> ligation--> transformation::
Digestion typically won't let you down. I would recommend doing a gel extraction directly after you have done your gel extraction. Keeping it in the at 4 or -20 iovernight will invite weak hydrogen bonding back on the fragments. However if you really need to go back home after digestion, trying heating the digestion reaction up to 65 for 10 min to break the weak hydrogen bond. It's not really pleasant to see additional band when doing gel extraction, you risk cutting out the wrong band.
Use TAE instead of TBE for subseqeunt reaction that requires ligation. Borate apparently inhibits ligation. And to decrease frying your DNA, you can put it on a gel tray then place on the UV box before viewing.
-Trim as much gel as possible front and back.
-regarding the PE buffer/ wash buffer, let them stand for 5 min as well b4 spinning them down, make ur stuff cleaner and "less salty"
- for good yield. resuspend EB buffer . Make sure it's heated to 65-70 degree, after pipeting on column let stand for 5 min b4 spinning them down.
I did 10 min at RT on cohesive end ( Ecor1) b4 --> fail @.@ but when i did overnight at 4 celcius, i got good stuff going. depending on the GC/AT of the end. if there're a number of AT overhang it would be better to do at lower temperature to promote their annealing.
Amount of ligase should be reduced. the stuff that u bought normally NEB for example..quite concentrated, dilute with 1x ligase buffer or so .
After ligation reaction, heat inactivate em at 65 for 20 min. for electroporation, dilute with TE buffer ( 5 fold) then use 1 ul to transform.
longer outgrowth period and less initial antibiotic conc. would certainly give less pressure to those cells .
that's what i can think of right now. If i miss out anything, feel free to add.
Cheers and happy new year may this topic brings success into your lab in 2008!!
During gel extraction reduce as much as possible or avoid DNA's exposure to UV.