I am creating an in situ hybridization probe and am linearizing 10ug of plasmid DNA containing my construct. Would you clean up the product before using it for T7 transcription or would you use it the way it is?
Many thanks!
Hi Monika,
Yes I routinely clean up the restriction digest done to linearize probe template prior to in vitro transcription. I usually cut 5-10ug of plasmid, run the digest over a spin column, and then elute with 20-30ul of elution buffer. This usually gives a template stock that is ~0.5ug/ul, convenient for setting up the transcription with. It's also a good idea to check the digest prior to moving forward with the transcription...
Cheers,
Ian
Hi Monika,
Yes, it is required to clean up the restriction digest, before continuing with the transcription reaction. The pH and salt concentration in the restriction digest reaction could be completely different from what is required in the transcription reaction, hence inhibiting the transcription reaction.
Best Simone
Dear Monica
yes as said in the past when I made probes for in vitro transcription for ISH like yourself I would always clean up
Dear Monica,
Yes, I do phenol/chloroform extraction and ethanol precipitaion, after the confirmation of linearization by electrophoresis. Then I dissolve DNA pellet with appropriate buffer.
Thank you all! I have done so and obtained around 200ng of linear DNA. I hope my transcription will be successful :) Thank you!
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