I have been trying to phosphorylate and ligate a blunt-ended linear plasmid after PCR. It remains linear. Any suggestions for troubleshooting?
I have tried 10 min at 70 degrees followed by ice for 2 min and then began the phosphorylation. Still only 1 band on my gel after the ligation reaction.
I have been following the protocol from: https://wellcomeopenresearch.org/articles/1-19/v1 , as this is the CRISPR plasmid I am using.
Any help is greatly appreciated! Thank you!
Was the PCR product amplified with Taq or a proof reading enzyme? Taq will leave an A-tail that will prevent blunt ligation-the A-tail can be removed by a short incubation with a proof-reader.
If you are gel purifying avoid UV light, switch to a 'Safe' stain and blue light.
Make sure that your ATP is fresh for the Kinase reaction (it is not present in some supplied reaction buffers and has to be added seperately).
Similarly with ligase buffers-they normally already contain ATP so aliquot them rather than freeze thaw a single tube multiple times.
You don't need to phosphorylate your insert, there should be a phosphate on the vector. Make sure the insert is actually blunt-ended, as Dr. Berrow said above.
Is your plasmid DNA a fresh preparation? Plasmids can degrade over time.
Thank you for your responses! It's much appreciated.
The PCR product was amplified with Phusion Polymerase. I've been using ligase buffer with ATP during phosphorylation. My plasmid DNA is new, I've only been using it a few weeks. I just switched to a new tube in case freeze-thawing is the issue.
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