I made different dilutions of control cDNA and in each case after running its PCR got the band of 496bp .The Gel pic is as below. But when I ran PCR for my gene of interest with same dilutions of cDNA (I extract RNA from plant and synthesised cDNA) as I made for control cDNA (whose RNA was in kit and synthesised its cDNA), the band which ought to be of 840bp was not obtained. The Gel picture of PCR for cDNA of my interest is given below. Total volume for PCR in both the cases was 50 microlitres. Please give as many suggestions as possible to optimize my PCR conditions for my gene of interest.
It is tough to tell from your method and your gel what exactly could be going on. But, some things to consider and look into:
1. Should your gene of interest (GOI) be expressed within this tissue and time point?
2. What was the quality of your RNA prior to cDNA synthesis?
3. How was the cDNA synthesized? Did you use an oligo-dT, random hexamer or specific primer during synthesis?
4. How were the PCR primers designed and did you check for secondary structures and hairpins?
5. Can you amplify your GOI from gDNA? The obvious thing that could be happening is your primers for your GOI are not specific.
That the control works only tells you that there is nothing wrong with the kit itself. Some possible causes/solutions to your problem:
- Your target RNA is not present in the starting RNA sample. Search the literature; is your target gene supposed to be transcribed under the conditions you are using?
- Poor quality RNA. Set up a control RT-PCR reaction for a target you know will be there, with a validated primer pair. If nothing shows up, there is probably something wrong with your RNA purification technique.
- Poor primer design. Use primers previously reported/validated in the literature. If your target shows up, then you know you have to change your primers and/or tweak their design.
- Non-optimal PCR conditions. Try a touchdown PCR, or if you have a gradient machine, try a gradient of annealing temperatures.
Hope that helps.
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