I have synthesised cDNA from plant RNA .The gel picture is given below. Please if anyone could tell me that whether the cDNA has synthesised or not? If synthesised, can I use this for Reverse Transcriptase PCR?
I have never run cDNA on a gel as a way to confirm cDNA synthesis. The easiest way would be to PCR it for an mRNA tht is expressed in your sample. Of course you need to be sure your original RNA is DNA free, maybe by doing on column DNAse digestion. All you will see on a gel is a smear and it is very difficult to know if this is RNA or DNA. Remember most cDNA reactions only copy RNA roughly 1:1 so you will not see much/any amplification here.
More info needed here: what are the lanes? Have you removed the RNA? Was the RT specifically primed or was it poly(dT) primed? You can always try the PCR and see what happens - Taq will pick up way, way less DNA than you will see on a gel.
hello, you can try one step RT-PCR kits on your RNAs, then run on the gel 1.5% (that is best)
Your gel shows there is some nucleic acid. in lanes 1, 3, 4 and 5. What it is is impossible to say. From random primers you will not get a single band on the gel so probably your cDNA has synthesised. It is possible what is staining on the gel is just RNA - it depends whether you removed the RNA before running the gel, where the RNA was from, how much you used in the RT, how pure it was, etc. I would say try the PCR.
I would only suggest that first go for the general PCR reaction with primers for constitutive genes(genes which always expressed or needed to survival for a cell) like actin gene. just run A simple PCR with Your Plant's actin Primers. if you are getting bands means you have successfully synthesized c-DNA.
All the Best
I have never run cDNA on a gel as a way to confirm cDNA synthesis. The easiest way would be to PCR it for an mRNA tht is expressed in your sample. Of course you need to be sure your original RNA is DNA free, maybe by doing on column DNAse digestion. All you will see on a gel is a smear and it is very difficult to know if this is RNA or DNA. Remember most cDNA reactions only copy RNA roughly 1:1 so you will not see much/any amplification here.
I don't know if your problem got solved or not. But I don't run cDNA on agarose, as I have read somewhere it will appear as a smear without any specific bands. I usually check the integrity of cDNA using plant 18S rRNA primers (5-CCA GGT CCA GAC ATA GTAAG-3; and 5-GTA CAA AGG GCA GGG ACG TA-3) which give a specific band at approximately 430 bp (+ PCR)
The smears that you see mean that your cDNA has been formed. What is the amount of cDNA that you have loaded? Did you performed a pre-treatment with RNase on the cDNA?
My colleague ask this question
I am working on cDNA-SCoT , but i got smear with the band
I would suggest you first amplify the cDNA by PCR. I amplified my cDNA by a normal PCR before running on 2% gel. Getting specific bands meaning cDNA synthesis is happening but still I get lot's of smears. Still looking for ways to obtain one clear band with no smears. Any suggestions?
I have never run cDNA on a gel as a way to confirm cDNA synthesis .
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