I already do the restriction process to the plasmid and the target genes. But, after it, I also do the inactivation of the restriction enzymes before I used it for gel purification. Now, I am preparing for their ligation process, but one question come to my head, did the inactivation of restriction enzymes to the plasmid and the target gene could be a problem for their next ligation process?
If you have heat inactivated the restriction enzymes, it does not cause any further complication in downstream processing. Make sure you purify the digested plasmid as well as the target genes by following gel purification &/or PCR clean up protocol. It removes all the Restriction Enzymes and buffers used for the restriction process so that it does not cause any problem when a second reaction (Ligation) takes place. Good luck.
Yes, certainly. If you carry active restriction enzyme oven into the ligation, it will chop your regenerated restriction sites, undoing the work of the ligase.
If you have gel purified your DNA, and it had exactly the expected size, you're most likely good. But if you have a small shifted band, it could be a sign that the nuclease is still stuck to it. Some enzymes stick harder than others.
If you have heat inactivated the restriction enzymes, it does not cause any further complication in downstream processing. Make sure you purify the digested plasmid as well as the target genes by following gel purification &/or PCR clean up protocol. It removes all the Restriction Enzymes and buffers used for the restriction process so that it does not cause any problem when a second reaction (Ligation) takes place. Good luck.
Hi there,
Inactivatin of RE is meant to avoid any interference with downstream steps.
hi Elly, just make sure that you CAN inacticate the RE. Some are heat resitent (like Taq I ;-) )
To all, thank you so much for your great answer. I am so happy to read it. Now, I am so confidence to continue the ligation process.
To Prof. Nellen: Hi Prof! How are you? Long time no see you. I heard from Siti that you are just back from German to Indonesia. Welcome in Indonesia, Prof!.. Thank you so much for your comment in my previous journal. Now, it has been published in RJPBCS and I already uploaded it in Research Gate. :-)
Hi,
Any clean up process (including gel purification) is most likely going to remove your enzymes whether they are inactivated or not so enzyme inactivation should make no difference to your ligations.
Are you visualizing with EtBr and UV when you are cutting from the gel? If you can change to a 'SAFE' stain and blue light instead you may get much better results, even a few seconds under UV can destroy your DNA and I know I take much longer than that dissecting bands from agarose gels.
Also check that your enzyme sites on the plasmid are not too close together if you are using more than one restriction enzyme.
To Nick S Berrow: thank you so much for your answer. Nice information. Yes, unfortunately, I do the visualizing with EtBr and UV when I am cutting it from the gel. May be it could be the reason why one of my gel purification did not show a result or a band. As my prediction, may be one of my gel purification result are in lower concentration or it could be like your explanation about the UV can destroy my DNA even for a few seconds.
Hi,
Your DNA should purify etc. OK but even after ligation the transformation efficiency of your product may be reduced after UV exposure. Concentrations etc. should be unaffected though.
If you are using a kit for gel extraction amke sure you follow the instructions regarding max gel weight, buffer pH and alcohol content of any wash buffers.
Remember to also have a molar excess of the insert to plasmid in your ligation mix and to dephosphorylate the plasmid if you are only using a single enzyme for linearization (or if everything you pick looks like plasmid only!).
To Nick S Berrow: thank you for your further explanation. Today, I already make the ligation cocktail for the transformation into E. coli that will be conducted for tomorrow.
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