While doing the PCR amplification of 16s rRNA gene of snail samples, I got a very nice pellet of DNA which dissolved in water readily. But when the PCR amplification was performed there appeared no bands on gel. When the nanodrop of the samples were done it was received like 1.11, 1.56, 1.8 etc. but no one amplified the gene. What could be the reason.?
in order to get a good amplification yes you need a DNA template of good quality; however little amount with the least acceptable quality of DNA can be enough for your amplification. here you say even at ratio of 260/280=1.8 you have not got amplification so it means that you have to look for the reason some where else. it can be because of many reasons relating primers, reaction reagents or PCR program. so with your provided information its difficult to find the reason.
Did you run a positive control (e.g. a sample that has worked befor or for other people) to make sure your PCR was working? Without a positive control such results/problems are almost impossible to solve. Just try to find a good postive control and run the PCR again...
Optimization of various reagents such as mgcl and tap poly may be needed and let see. Be clear that much can lead to much noise/background
Try diluting your DNA from 1/10 to 10(-3). This may help in reducing the contaminants.
What was the concentration of your DNA on the NanoDrop reading? I tend to find that more useful than the 260:280 ratio. I find that if my DNA concentration is low, my 260:280 will be low as well. I design my PCR to use 30-50ng of DNA or increase the number of cycles if I use less. I do agree with the others that you should use a positive control as well to help identify any other problems that may be occurring.
Thanks every one for the reply
In the next set, I am trying out with positive control as well as serial dilution of DNA to prevent inhibitors. I have been using the same reagents and primers for a long time but only recently the problem arose. I am using the Phenol/Chloroform extraction method. The nanodrop concentrations range from 100 to 200 ng/ul.
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