extracted DNA from soil, afterward I have run PCR using ITS primers but did not get any band in the gel what could be the reason??/
Without a control bacterial DNA (positive ctrl) it is difficult to answer! Have you seen the primers on the gel?
At this point, you are troubleshooting, the electrophoresis, the PCR and the DNA Isolation so this needs to be done in a step-wise manner. The easiest to eliminate is the electrophoresis technique: if you see your ladder separating as it should, the electrophoresis worked.
If so, did you include a positive control? Did it amplify? If yes, the problem is with the DNA isolation (either presence of inhibitors or no DNA isolated). If no, then the PCR has issues too, on a more basic level (you need to optimise the primes....after making sure that the primers themselves are not degraded).
Once you eliminate the PCR as having worked well...then trouble shoot the DNA isolation.
Without checks and controls as mentioned above by both Herve and Bhaskar, it is impossible to know where the problem is. You need to eliminate possibilities one at a time.
Just to add to the excellent suggestions above, what is your target (fungal?) and what primers are you using? What extraction method did you use on the soil? These will all impact on the experimental outcomes.
Above all you need to remember that in soil can be a lot of ihibitors of the PCR. If electrophoresis works (ladder is seen) you may need to purify your samples.
I am agreeing with all of the above mentioned points. Soil can be a tricky subject for nucleic acid extractions. Make sure you have high quality DNA to work with by using the nanodrop, Qubit and for higher concentrations you can even check the integrity with a simple agarose gel. If inhibitors are present in the sample (check your A260/A280 and A260/A230 ratios) you can use spin columns to remove most of them.
The primers need to be tested in advance and a positive (as well as a negative) control should be included in your experiment, for sure!
Another detail should be the DNA concentration used in the PCR. Maybe try different dilutions. Furthermore, you could add PCR enhancers such as DMSO or increase the MgCl2 concentration. The choice of polymerase and temperature settings are also very important for a successful PCR.
- Badges
- Science method