Hello academics
I have a question regarding standard PCR. These are my components for PCR
cDNA 1microL
F primer 1
R primer 1
dNTP 2
10x buffer (with Mg+) 2.5
ABM taq 0.25
DW 17.25
Total volume: 25
Despite carrying out PCR a number of times along with different primers and taq (Takara), I get no bands in the gel. Hence am starting to assume that probably my cDNA is the problem here. I extracted my RNA using the trizol method (did not use DNAse treatment) and converted it to cDNA using the power cDNA synthesis kit and then did further 1/20 dilution of the stock cDNA.
My question is, is there an ideal concentration of cDNA (ng/uL) required for PCR or is there something I may be overlooking? Any form of help would be much appreciated.
Thank you in advance
Dee
Hi,
Well, unfortunately, there could be hundreds of different reasons that would cause no bands after PCR. If you are sure that the polymerase, all reagents, and primers are fine and should be working, then possible problems with cDNA are as follows: 1) you extracted the RNA with Trizol, I understand that the washing step was with ethanol, right? Are you absolutely sure that the ethanol evaporated completely before reverse transcription? The contamination of RNA with ethanol will inhibit the reverse transcriptase, and you will not see it on a spectrometer 2) What concentration of cDNA did you synthesize? 1.5ug/ul? 3) are you sure that you did not let the RNA degrade during the extraction? From what kind of material are you extracting? How did you homogenize the tissues? In terms of cDNA concentration - it depends on the manufacturer of your PCR kit. Also, do you have just one sample that worked in the past? For example, from a previous experiment or another friend in the lab? With this kind of "positive control," you could check if the problem is with your reagents (If the positive control does not work) or if the problem is with your cDNA. If its cDNA, it would be a good idea to extract just one sample from fresh material to check if the reverse transcriptase is working. What about the primers - are you sure they are not contaminated or not old? For which genes? Are those genes abundant for all samples (like 18S, 16S, ITS, actin, 28S, etc.), or is their expression dependant on your treatment of choice? In this case, are you sure if they were expressed at all? Perhaps you could also extract the whole DNA instead of RNA just to see.
I hope it will help you. Don't worry - it happens very often, you just need to check a few things, and by doing that, you will start to limit the possible reasons behind the empty gel. Good luck!
Also, check your
RNA quality, 260/280 ratio; PCR cycle
Use a positive control & make sure you have correct primers
Dear Depika,
During setting up the reaction , please try to have a positive control, negative control and a no template control, that would confirm you that your pcr ingredients are working properly and if there is any problem with the cDNA that you are using. If evrything remains ok in the control sets then u play with the annealing temp. And conc. of template. But i would recommend you to prepare the cDNA freshly.
Hi! I would suggest to adjust the conditions of amplification with DNA. When you have good amplification with DNA you can start to test your cDNA. As it was mentioned before, there are several steps that can be failing from RNA extraction to cDNA synthesis. I would suggest that you check that your cDNA is ok by amplifying a housekeeping genes as these are likely to be expressed across your samples. It also can be that your target gene has low expression in your samples... so you may also try to work with less diluted cDNA or even the total cDNA to see if the problem is of detection.
Good luck
Hi Dee,
this is a good paper for PCR optimization:
Article Multiplex PCR: Critical Parameters and Step-by-Step Protocol
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