I am generating RNA probes for in situ hybridization in mussel mantle tissue and am having poor DIG-labeling efficiency. I extracted the RNA using the RNeasy Mini Kit and had good quality RNA. I synthesized the cDNA probe templates via 2-step RT-PCR (first strand synthesis using oligo d (T) primers to generate a pool of cDNA, followed by PCR with target specific sense and antisense primers with a T7 promotor. PCR products were cleaned up using EXOSAP-IT and then used in the transcription and labeling reaction (Roche DIG-RNA labeling kit). I ran a kit control and it worked fine, however my probe bands were very weak. I know that the purity of the DNA is crucial for transcription reaction. Should I be using a different method for post-PCR cleanup?
I do not know the cause of your poor probe production but I will caution you about one aspect of exo-sap purification.. Exo-sap does not degrade double stranded dna ( which is why you are using it to eliminate primers) but that aspect can,itself, cause a problem. If the primers can self anneal as in G quadruplex or loop formation due to self complementation or can anneal to some sequence in the other primer ( high gc complementarity) then we have double stranded dna present which will not be nucleased and are not part of your expected target dna. If this is likely to cause problems either in the quantification of the dna present or in the later reactions then I would change the purification method
Laurence Stuart Dawkins-Hall
and Paul Rutland , thank you for your insights. I am going to try the Roche High Pure PCR Product Purification kit.
It would be interesting to know how the new cleanup method works for you Kimberley Hobbs
good luck
Paul Rutland , Thank you. I will update my post after I try the new method.
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