More PCR cloning problems! - (Dec/06/2006 )
By PNK do you mean polynucleotide kinase?
If so, is there a way of getting unidirectional concatamers??
thanks.
QUOTE (perneseblue @ Dec 8 2006, 03:34 PM)
set up a quick ligation and add 0.5ul PNK. (If your reaction volume is more then 40ul add 1ul PNK) Incubate for about 2hrs at room temperature ~25 Celcius.
After that, you can check if the ligation has worked, by taking an aliquote and running said aliquote on a gel. You can heat kill it first if you want, before running the gel.
After that, you can check if the ligation has worked, by taking an aliquote and running said aliquote on a gel. You can heat kill it first if you want, before running the gel.
-brami-
yes; PNK = polynucleotide kinase
In this situation directional concatemers is not requried. As in the next step you are going to digest the concatemeres with NdeI and BamHI, breaking said concatemers back into monomer (with overhangs now).
-perneseblue-
Yes, you are right.
But I am interested in making unidirectional repeats of a 100bp PCR product and use this concatamer of linear tandem repeats for transient transfection for expression in mammalian cells.
Apologies if you consider my posting an additional question within a thread, as inappropriate.
thanks again.
QUOTE (perneseblue @ Dec 8 2006, 03:49 PM)
yes; PNK = polynucleotide kinase
In this situation directional concatemers is not requried. As in the next step you are going to digest the concatemeres with NdeI and BamHI, breaking said concatemers back into monomer (with overhangs now).
In this situation directional concatemers is not requried. As in the next step you are going to digest the concatemeres with NdeI and BamHI, breaking said concatemers back into monomer (with overhangs now).
-brami-