Heating the vector and insert before ligation - new idea for reducing background colonies (Feb/21/2007 )
Hi all,
Unfortunately, I often have to clone into a single EcoRI site and had a thought today that a good way of reducing vector re-ligation was to heat the vector preparation or an aliquot of it at 37C for 2 minutes before I used it in the ligation. My theory was that because some of the 4 bp overhangs on each end of the vector may anneal to each other (forming a temporary circular vector), a little heat would denature that 4 bp stretch and prevent what would be a pretty simple ligation from occurring. The same could be done for any insert with an overhang to prevent it from ligating with other inserts at the same restriction site. What do you guys think about this? Do you think a little heat could be helpful?
Cheers, Rob
Unfortunately, I often have to clone into a single EcoRI site and had a thought today that a good way of reducing vector re-ligation was to heat the vector preparation or an aliquot of it at 37C for 2 minutes before I used it in the ligation. My theory was that because some of the 4 bp overhangs on each end of the vector may anneal to each other (forming a temporary circular vector), a little heat would denature that 4 bp stretch and prevent what would be a pretty simple ligation from occurring. The same could be done for any insert with an overhang to prevent it from ligating with other inserts at the same restriction site. What do you guys think about this? Do you think a little heat could be helpful?
Cheers, Rob
hi,
do you mean that you want to prevent self-ligation?
if so, I did it before.
at the time, I used BAP (bacterial alkalin phosphatase) to prevent self-ligation.
Then, after BAP treatment, i used PCR clean-up kit from Qiagen to remove BAP residue.
Oh yeah sure, de-phosphorylation is a given. I use SAP, isn't 100% efficient though. Still some background colonies.
hi, i have heard about this method too and have used it. mmm...didnt notice any difference, but why not try?
Oh ok. Figured it had been tried before elsewhere. Yeah I had my doubts, I suspect the temperature at room temp could even be enough to keep them apart but thought I'd put it out there and see what people thought any way. I screen my results tomorrow.
hi
i do slight different. After purifing vector and insert, i put them + water in a tube, and heat both 2' too.
tend to enhance insert ends disponibility.
but four tough cloning, i dindn't see radical effect:(
i do slight different. After purifing vector and insert, i put them + water in a tube, and heat both 2' too.
tend to enhance insert ends disponibility.
but four tough cloning, i dindn't see radical effect:(
hi fred_33 can you please mention the temperature and time of heating??? i want to try it in near future
thank you
I heat the insert and fragment for 2-3 min in a 65C block before adding them in the ligation mixture. i am not sure if it really helps or just some voodoo. But anyway I heat them.