I have been trying to run agarose gel electrophoresis for some days but not successful. I initially used RNA, convert it to cDNA and run PCR reaction before using for electrophoresis but only the ladder appears. Later, I extracted gDNA and add Master mix; run the PCR Reaction before running electrophoresis. Again, only the ladder appears. What can I do and what am I doing wrong? I ran electrophoresis for the integrity of the RNA and gDNA, they are present in the extracted samples
Hi there,
It seems that your RT-PCR failed. So check the protocol carefully and run a control in parallel to prove the system is working fine with control RNA and control set of primers.
Have you check the RNA and gDNA yield before you run the PCR.
If yes, I do expect that there is faulty in either the master mix or the protocol used.
It can affected by the optimum annealing temperature or the primer used.
Hope this help.
if your primers are correctly designed then the product from genomic dna is probably too large to amplify so no problem there. Dominique is right. You need a housekeeping gene primer set which will amplify from your cdna to show that you do have some cdna otherwise you will not know if there is no cdna so no amplification or if the cdna is ok and your primers do not work because of the pcr conditions, Ask around your department for primer sets that have worked with other peoples cdna. beta actin is an often used control gene primer set
I used beta actin as my control gene. I am going to look at the cDNA process or the PCR Reaction maybe the annealing temperature was not right
You can check gDNA on agarose first, don't trust nanodrop results.
Therefore, you can add positive control with your samples.
But you have to check the PCR program well. I guess you can prolong the extension time to be 1m or more. Good Luck
it can be more useful if you can upload some pictures of your unsuccessful results , they are a lot of reasons behind that problem
Check steps...Is ur primer is correct, and try with the primer pair for other gene with same cDNA. Then check ur Master mix. one more thing is also posible the ur gene amplify in specific conditions or treatment.
After checking ur RNA, u should perform DNase treatment to remove DNA contamination. Then perform cDNA synthesis, all components thaw in ice. brief centrifuge them before using. mix enzyme very well. all these processes should be completed within a day. Check ur annealing temperature.
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