I will be procedding with rna probe designing.
but before that I am performing pcr to create antisense and sense products and then purifying them. I have performed PCR and using QIAGEN PCR purification kit, I purified the pcr products (plasmid dna with gene) and measured its concentration (in all I had 4 samples for one gene 1antisense and 1 sense respectively). Also please note for each antisense and sense I had 3 pcr reactions which I pooled them in the end and then proceeded with PCR purification. The concentrations are 61.6ng/ul, 10.3ng/ul, 14.1ng/ul and 11.3/ul. I need final concentration of 1ug of each to proceed further.
Do you know how to improve the yeild or any other method to increase concentration.
Please advise. I would appreciate if anyone can tell me how to go about or proceed.
This subject has been discussed several times on RG so far. Here is a thread with great tips:
https://www.researchgate.net/post/PCR_purification_problem2
I have made many RNA probes, and I don't use a kit for purifying the plasmid DNA. I do a dirty miniprep, basically P1, P2, P3, and then phenol chloroform, followed by EtOH precipitation. It's cheaper to do it this way and also you get higher yield. In my protocol, the probe is purified during the final step when cleaning up the RNA, so it is not necessary to keep in clean in earlier steps. My probes have always been good. I can send a protocol if you want.
Yes Liz Unger It would be of great help if you could send your protocol!
But one thing I didnt understand dirty miniprep? :P please explain.
^^ see we run pcr for each probe reqction (antiwsense and sense) using m13 F or m13 R right along with the either or rev primers of gene of interest. so after this (pcr) you pruify the template inorder to use for the transcription reaction right?? so whioch kit did youu use for purification(qiagen PCR purification kit or any other method)
# also, how to get 1 microgram of DNA template for RNA synthesis (rna probe preparation) ? i have tried to run 3 PCR reactions, 50 ul each, but after then purification with Qiagen PCR kit, I gets sometime very low concentration. How did you do before RNA probe synthesis?
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