PCR bands on gel electrophoresis - (Mar/23/2010 )

HI everyone!


I had a new problem in my PCR. I have run the PCR for 5 samples as a trial. Then when i run the gel, i can see very nice and clear bands. When i run back the PCR for my 45 samples, i cant see any bands on the gel. I did twice, but still no bands. Anyone have any idea on this, why i got no bands for PCR products? I used the samples for my trial and i followed all other procedures exactly the same!

Looking forward for reply as soon as possible!

Thank you!

Did u run a positive control?

You can use the same sample that yield a product in the first time, and using that as a positive control for the other 45 samples. If your positive control did not show up now, maybe it is because of your PCR settings, or during the preparation step, did u change any reagents? Did u store your DNA properly?

Maybe try again..

I used the same samples (5 samples) that yield a product in the first time. Now i run the PCR for another 40 together with that 5 samples earlier i used. The problem is now there are not even a single bands is visible for those 45 samples. The PCR settings is exactly the same with earlier settings. During the preparation step also, i did in sterile condition and accurate pippette techniques! I didnt change any reagents. The buffer, primer, dNTP, Taq, MgCl2 all the same with the earlier.
Yes, i do store my DNA properly. I stored it -20 degree celcius.
I'm really confused about the problem. I am a final year student and this problem occur on my final year project. Now for my project paper, i need to discuss why i didnt get the bands! I dont know how and what to explain this!
Please anyone could help me?

Looking forward for reply as soon as possible!

Thank you!

How many times have you tried to repeat the PCR?

You should always use a positive control in your PCR. There could be plenty of reasons why it didn't work, but you can not know for sure unless you have a positive control.

Maybe try to repeat PCR again?

Another reason I can think of is if your DNA concentration is low.. how about increasing your DNA template? when you take your dna out from -20 celcius, did u mix it before u pipette out your DNA?