Hi all.
Im trying to clone a purified PCR product (450 bp) into pBSKS with SmaI digest, first I tried doing all at the same time at 4ºC resulting no one white colony, and then I tried doing first the digest at 25ºC around 4h and add ligase for over night at 4ºC.
Im using all from thermo.
Every suggestion is welcome.
Thanks!!
Using PEG4000 should enhance this kind of ligation. Also keep in mind that if you are ligating such a small piece it might ligate in frame with lacZ alpha and not disrupt its function therefore checking some of the blue colonies might be necessary.
I would dephosphorylate the vector with CIP after SmaI digest, phosphorylate your PCR primers if you have T4 PNK around then redo the PCR, and gel extract the linear plasmid after SmaI digest and do the ligation with those. It's a much more likely reaction for the vector to reclose than it is for two separate blunt ended pieces to meet and be ligated into a closed circle if the vector isn't prevented from self ligating, plus there's always some small amount of DNA that doesn't get cut by the enzyme and the combination of those two will add a ton of background to your ligation.
Alternatively you could just redesign the primers to add two useable sticky ends, that's probably easier and cheaper.
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