Problem with the PCR Product and Extraction - (Aug/13/2006 )

Hello,

I had my first PCR experience today. I did it alone following certain written procedures in the lab. HOwever, I think that I am not sucessful this time. Attached is the gel electrophoresis image of my 4 samples.

Here is my procedure;

１． 3 mL of 10^9 cells/ml of bacterial sample was centrifuged for 10 min at 12000 rpm
２． 40 µl of sterile Milli Q was added to the pelleted cells and stirred
３． Freeze―thawing was done twice ( freeze for 10 min at -20 C and thawing at 60 C for 5 min)
４． 10 µl of 1mg/ml Proteinase K was added and vortexed for 1 min.
５． 50 µl TTNE Buffer was added and vortexed for 1 min.
６． 60 C for 20 min (Enzyme Reaction)
７． 95 C for 10 min　（Deexcitation）
８． Centrifugation at 14000 rpm for 5 min and supernatant was collected.

100 µl PCR reaction was carried out using the following

0.5 µl Ex Taq
0.5 µl of primer 10 f
0.5 µl of primer 1500 r
10 µl of 10xEx Taq Buffer
8 µl of dNTP mix
5 µl of 100 mM MgCl2
2 µl DMSO
2 µl of the supernatant sample
71.5 ml of steriled Milli Q

PCR condition:35Cycle

Preheat 95 9min
　 　　 　 95 30sec
50 30sec
72 1min
4　　∞


I used λ/Hind III as a marker. Could you plese help me identify to what portion should I have to improve next time. What could be the possible reasons? Have I failed with the extraction method or PCR reagent composition/PCR condition? Thank you in advance for your help

All the 4 samples look degraded as you can see long smears instead of proper bands. Make sure you don`t have any nucleases in your reagents. Maybe repeat your PCR and run your samples immediately afterwards. Use fresh running buffer in the gel electrophoresis chamber.
Check whether you see your desired fragments, no fragments at all or unspecific amplification. According to this you might have to optimize your PCR.

Hello,
i seems to me that the PCR did not work.so positive and negative controls r necssary when u do PCR.

i think 9 min by 95° will destroy some of the Taq if u do not use Hot Start. i generally use 2-3 min with the presence of DMSO.

i usually use 5% DMSO and not 2%...

but before u change all this just do a positive control with the same condition...

Good Luck