RT / cDNA synthesis - (Jul/03/2012 )
Hello everyone! This is my first time performing RT for the purpose of synthesizing cDNA, and I had a question for some more experienced hands.
I was wondering if it's possible to use the same primer during first-strand synthesis and the subsequent PCR for amplification.
For example: If I were to use an oligo dT primer to capture an mRNA, would I be able to use the dT oligo during the PCR amplification? I understand that the dT sequence is technically (-)ssDNA (same as the ss cDNA made during RT), so it wouldn't bind to the template used in the PCR. However, is it possible to allow the first extension step to run for an extended period, allowing the polymerase to synthesize the full (+)-sense cDNA and THEN allow for the dT primer to bind and syntehezie an entire (-)-sense cDNA? I think that would allow for subsequent amplification in the later PCR cycles. I guess this would be like synthesizing two strands of cDNA on the first step in sequential order.
Any thoughts?
You could, but oligo dT is very non-specific so you would get a massive amount of competing reactions that would make your PCR very very inefficient.
Thank you, bob1, for your reply.
I realized the answer to my question about halfway through the PCR cycling (haha). I'm actually using gene specific primers, but I see your point about the oligo dT's. Anyway, the reaction worked nicely and I'm about to transform the PCR product into E. coli. Thanks again!
LabLackey on Tue Jul 3 16:04:39 2012 said:
Hello everyone! This is my first time performing RT for the purpose of synthesizing cDNA, and I had a question for some more experienced hands.
I was wondering if it's possible to use the same primer during first-strand synthesis and the subsequent PCR for amplification.
For example: If I were to use an oligo dT primer to capture an mRNA, would I be able to use the dT oligo during the PCR amplification? I understand that the dT sequence is technically (-)ssDNA (same as the ss cDNA made during RT), so it wouldn't bind to the template used in the PCR. However, is it possible to allow the first extension step to run for an extended period, allowing the polymerase to synthesize the full (+)-sense cDNA and THEN allow for the dT primer to bind and syntehezie an entire (-)-sense cDNA? I think that would allow for subsequent amplification in the later PCR cycles. I guess this would be like synthesizing two strands of cDNA on the first step in sequential order.
Any thoughts?
I want to know how did you deal with the DNA contamination in the RNA sample, or they are not going to affect the result ?
thanks
volans1900 on Thu Jul 12 12:59:11 2012 said:
I want to know how did you deal with the DNA contamination in the RNA sample, or they are not going to affect the result ?
thanks
You add DNase to the isolated RNA, then remove the DNase and finally synthesize the cDNA....
bob1 on Thu Jul 12 21:09:06 2012 said:
volans1900 on Thu Jul 12 12:59:11 2012 said:
I want to know how did you deal with the DNA contamination in the RNA sample, or they are not going to affect the result ?
thanks
You add DNase to the isolated RNA, then remove the DNase and finally synthesize the cDNA....
I did that, but after run for RT+ and RT- , I got non band for both ... seems either I have RNase contamination or the heating inactivation process degrade the mRNA.
I think change primers to make DNA amplification product larger than cDNA is better ...
I am using (RNase Free) DNase I provided by Ambion, and the heat inactivation process is 75 Celsius for 5 min. I did add EDTA to 5mM as instructed to prevent RNA degradation. as for the Mg2+ concentration, can I add the DNase Buffer, since it contain MgCl2 inside..
Check your primers and make sure you have a positive control (perhaps a plasmid with the cloned gene) before you suspect the RNA.