6 replies to this topic

[Vee OS]

Master mix
dNTPs (0.5 µL)
1 x buffer
Primer 1 (T7) (0.5 µL)
Primer 2 (SP6) (0.5 µL)
Taq polymerase (1 U)
Template DNA (1 µL)
Top up to 25 µL with deionized H2O

PCR parameter
94C - 5mins (Initial denaturing)
*94C - 0.5min
*45C - 0.5min
*72C - 0.5min
72C - 10min (final extension)

*Repeats 30 X

Electrophoresis process (45mins at 90V)
Marker DNA 100bp (0.2 µg ladder)
PCR product should be ~550bp

My problem
Why does my gel yields three bands? Is it something wrong with my procedures? The other two fragments are bigger than the last band. Why is it so?
I can't figure it out. Please help. Thanks in advance.

[vladooo]

It might be non-specific primer binding. Increase the annealing temperature in 5°C steps. 45°C is very low.

[Adrian K]

What Taq are you using?

[dreww]

like above: non specific binding, but what are you PCRing? could be genomic or plasmid, or both.

lanes 3 and 4 have either nonspecific binding, or contamination.  whats your source of deionized water? have you tried nuclease free H2O?  could be contamination from another pcr or what not. try re pcr it with difference controls. i.e. no template control, change H2O source, etc.

Edited by dreww, 05 April 2010 - 02:36 AM.

[tea-test]

is it possible that those upper bands are not amplification products but your template plasmid? they are much larger (several kb?) than your pcr product and i don't think that the polymerase is able to amplify such large fragments in just 30sec.

how much template (ng) do you use?

[dreww]

tea-test, on Apr 5 2010, 01:47 AM, said:

is it possible that those upper bands are not amplification products but your template plasmid? they are much larger (several kb?) than your pcr product and i don't think that the polymerase is able to amplify such large fragments in just 30sec.

how much template (ng) do you use?

thats what i was trying to say.  thats probably what it is.  probably was a "dirty" pcr vs a purified fragment

[Vee OS]

Thank you so much for your advises. Been busy with hectic schedules but I did read you guys' comments. No time to reply though. I find out what's wrong with it after all. The master mix was prepared by my partner. She contaminated the mix while preparing it. Well, everyone knows we are forbid to talk especially during PCR experiment. She talked in infront of the microcentrifuge =.=! Those bands are contaminations...

Anyways, thank you so much! I've submitted my report to my lecturer. ^^

Best regards,
Vee OS