I created sense and antisense probe by extracting and purifying RNA, generating cDNA, performing PCR for wished construct, which I then cloned into pCR-Blunt II - TOPO vector. After isolating and purifying the plasmid, I linearised it so I could use either SP6 or T7 promoter for RNA polymerase to generate sense and antisense probe. The RNA transcript was purified, DIG-labelled and quantified on a QUBIT. After this procedure I only obtained the concentration of about 300 ng/ul.
How do I get at least 1 ug/ul and how do I dilute my probe in hybridization buffer so I have sufficient concentration? I am trying 30ng/ul at the moment.
Thank you for all your answers!
Hi Lale!
Thank you for your answer! I think I'm a bit paranoid when it comes to RNA, so I clean up after every possible step :) I did everything the same but the precipitation.
This is the first try, so I hope I'll get a signal at least from my control probe :)
Hi Monika,
Don't know about the protocol you're using. I used the LiCl precipitation method and it worked well for me.
I also used to double the volume of the reaction (so 40 ul instead of 20ul, double all ingredients) and keep the reaction for 4 hours before doing the precipitation. In addition, I also used Glycol Blue to help me see the pellet so I didn't lose any while discarding the supernatant.
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