very important - electrophoresis problem (Nov/16/2008 )
Hello every body,
I need help........
I do colony PCR with PFu polymeraze enzyme & then agarose gel electrophoresis for the product & I repeated my work many times in which i got results twice but other times i got strange results (primer dimer only on gel or no bands at all).
Putting in consideration that i prepared my TAE buffer & also my loading buffer myself.
I don't know what is the problem, i prepare 1.5% agarose gel with 0.5% ethidium bromide & run them on 120-150 volts with 200 Ampere.
do you think there is a problem in PH or buffer reagents??
Isn't the problem more likely to be due to the PCR conditions than the electrophoresis conditions?
No it is not because it gave me results twice.
Did you have a marker that ran correctly ? If marker did run correctly then doubtful its an electrphoresis problem. If you didnt run a marker try running one with your reagents and running one with someone elses.
I guess I can't understand how electorphoresis conditions could possibly be the cause of "primer dimer only on gel or no bands at all"...
I agree with hombrew. No bands or primer dimers are not likely to be a gel problem, but a PCR reaction problem.
I need help........
I do colony PCR with PFu polymeraze enzyme & then agarose gel electrophoresis for the product & I repeated my work many times in which i got results twice but other times i got strange results (primer dimer only on gel or no bands at all).
Putting in consideration that i prepared my TAE buffer & also my loading buffer myself.
I don't know what is the problem, i prepare 1.5% agarose gel with 0.5% ethidium bromide & run them on 120-150 volts with 200 Ampere.
do you think there is a problem in PH or buffer reagents??
What are your PCR conditions? How much colony do you add? Do you do a positive control with purified DNA?
I need help........
I do colony PCR with PFu polymeraze enzyme & then agarose gel electrophoresis for the product & I repeated my work many times in which i got results twice but other times i got strange results (primer dimer only on gel or no bands at all).
Putting in consideration that i prepared my TAE buffer & also my loading buffer myself.
I don't know what is the problem, i prepare 1.5% agarose gel with 0.5% ethidium bromide & run them on 120-150 volts with 200 Ampere.
do you think there is a problem in PH or buffer reagents??
What are your PCR conditions? How much colony do you add? Do you do a positive control with purified DNA?
I don't see why it could be an electrophersis problem
very likely a PCR problem
and it's not uncommon that you cannot repeat your results when you got it right twice
It's very possible that the PCR condition you are using is sub-optimal, and some minor variations between experiments occassionally gives you a band.
In fact this is quite likely, given that it's colony PCR -- quite possibly the dirtiest of all, and prone to false negatives. Swanny's suggestion of trying the PCR with extracted and purified DNA would go a long way towards sorting out the real issue here...
Thanks a lot to everyone who tried to help me
I finally discovered the error. It was the water!!
YES, there was a problem in the dissilator so PH of water was not controlled.
But now i repeated my work again & there is another problem...
bands faint quickly before the marker separates well
so that to get good obvious bands, marker is not well separated yet & needs more time,
but if i left it for more time, bands faints !!
I don't know how to fix that. Can the problem be the agar type or what???