Hi,

did you vortex your sample before you did the second PCR? How many µl DNA are you using for your PCR? How often did you repeat your experiment?

Cheers, Nadine

It is helpful to list your PCR conditions and the recipe for your PCR solution.

Some plants are high in compounds that may inhibit enzymatic reactions, such as PCR. You can check for the presence of inhibitors in your DNA preps by diluting the sample (1:100 and 1:1000) and running 36-40 cycles of PCR. If the dilutions work better than your undiluted sample, you may have PCR inhibitors in your DNA preps.

Hi

yes I vortexed the sample before use usually am taking 2µl of DNA what ever concentrations i use for the first time i am using the same for second time also

I observed that when I increased the concentration of DNA am getting the fiant bands but earlier with less concentration only it was amplyfying how can be possible

Assay buffer:2µl

dntps: 2µl

Taq: 0.8µl (3u/µl )

f.p:1µl

r.p:1µl

tmplt:2µl

this is what am using

Too high DNA-concentration can also inhibit a PCR reaction!

Using too much total DNA results in packed DNA in the confined space of the reaction vessel and can lead to false priming and even poor DNA synthesis due to the obstructed diffusion of large Taq polymerase molecules. However the ratio of target DNA to burden DNA is also important.

@Maruf: Without any information about your used concentrations eg. primer your comment isn't helpful! Cheers, Nadine

@Maruf: Then it will be nice to name us/her the publications!

Thanq verymuch for all ur responses i will try wth diffrnt cmbinations and ill post my result again

Hi Bharathi,

Sorry if I am a little late with my response. I do have one question to consider. When you stated that you had amplification the first time, how much amplification did you have? Was it just enough to observe on a gel or a very strong band? If you only had a light observable band, there may be very little difference between an observable amplification (first time) and amplification below ability to detect (second run with more template DNA). I would agree with Nadine that an increase in template DNA could inhibit amplification. This could be the difference between whether you had a relatively strong band or no band at all. However, if the successful amplification was very light, a third run of PCR may or may not be successful because the PCR may need further optimization. Addition of Betaine or DMSO can also be helpful. Please let us know how it turns out.

Thank u for ur response Bintzler

But for the first time I got a very strong band for second time some times am getting Faint band if it is further repeated again no band was observed

What you are describing in your PCR sounds like possible degradation. The question would be degradation of what in the reaction. I would generally not believe primers degrade very quickly. So, it may be worth looking at the template and polymerase as the most possible suspect. If not template or polymerase, then you should look at the other reagents in the PCR. First question is how did you isolate the template. Was it by using a kit like Qiagen or was is a resin based kit. Years ago people were using resin based kits and some of the resin was still getting into the final product and eventually reabsorbed the DNA. We saw this problem a lot in sequencing. It is also possible the template is degrading. Has the template been undergoing through freeze-thaw periods. Is it stored in buffer or water? Either way I would quantify the original template to be certain of the concentration. You may also want to check the polymerase. Have you included any control samples? Perhaps you could try re-amplifying the original PCR that worked well if you still have some. If you are by chance making a master mix with dNTPs, buffer and polymerase, I would not recommend vortexing because this could denature the enzyme.

So, I would look at the template and polymerase first. If you are still having problems then please let me know. And also let me know if it all works out.

Take care,

Doug

Thank you very much for all your responses I had tried PCR by diluting DNA and I got the result as expected once again Thank you very much