hello friends
recently we have got StrataClone PCR cloning kit to clone a gene of interest about 6.1 kb size.
I used LA Taq DNA polymerase for PCR, Gel purified the bands, incubated it with Taq DNA polymerase and dNTP at 72*C for 15 mins so as to incorporate 3' A overhangs.
Later following the mentioned protocol, added 3uL of buffer+ 2 uL insert+ 1uL vector mix9Incubated 10mins at R.T). Transformation was carried out with provided competent cells producing Cre recombinase.
Plated 3uL on LA+Amp with 40ul 2% Blue-gal, also plated 3uL on LA+Amp.
But after overnight incubation nothing appeared. Not even circular nothing.
Kindly suggest me a way to get the clone.
So you are saying you don't get any colonies at all? Even if the cloning of the insert didn't work you should see blue colonies. This means (a) something is wrong with your cells. Did you do a control transformation with the provided pUC18 plasmid? Or (b) something is wrong with your plates.
By the way, according to the manual you should use 5–50 ng of your PCR product for the ligation step, which equals typically a 1:10 dilution of a robust PCR reaction. You didn't mention it so I just wanted to make sure you didn't miss this step.
Forget the second part of my answer. You use gel purified insert and therefore shouldn't dilute according to manual.
Hi Cornelia
i used 120ng/ul conc. of my gel purified PCR product. And yes, i think you are correct, the competent cells may some issues.
Should i test the transformation efficiency only with the pUC18 or should i perform cloning with the provided insert as well?
I would start with the transformation efficiency. After you ruled out problems with your cells move on to the ligation part. You need to transform the ligation product therefore make sure first your cells are ok.
Hi Cornelia
Thanks for the advice. I tested the provided cells with 10pg of pUC18 and they lack competency .
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