Unexpected bands for negative controls of my PCR on the gel

[lisalin]

Hi all, recently I came across a repeating problem that the negative controls of my PCR always showed bands on the agarose gel.

When encountering this for the first time, I ran another PCR trying to figure out what was/were contaminated, water, mastermix or primers? But again, even the PCR product generated from the mixture of newly bought mastermix, water and primers showed bands on the gel, urrh actually, two bands on the gel (around 80 bp and 200 bp, respectively). I have no idea what was going on with my PCR operation--I even performed the preparation of my PCR in a laminar flow hood, and disinfect everything including my gloves several times when there was any trace of contamination. Would it be possible that the filter tips were contaminated? Or else, the formation of primer-dimers due to inappropriate temperature? Help!!! Please....

[Aga]

I would autoclave the pipettes first - they tend to suck up some of the liquid and may be a source of contamination.
If you use sterile pipette tips (but not reused) they should be fine. Have you tried to change each of the reagents?

80bp fragment for primer dimers is a little bit too long, so it's rather something else.

[butters]

is the unwanted band as intense as ur positive control? or very faint but slowly increasing when more pcr is perform? where u load ur DNA?

[lisalin]

Originally Posted by butters

is the unwanted band as intense as ur positive control? or very faint but slowly increasing when more pcr is perform? where u load ur DNA?

Actually, the unwanted bands were a bit faint, not as intense as my positive control; also, the banding patterns between these two were slightly different. I loaded the PCR products with template DNA into the wells beside my positive control on the gel.

[butters]

actually what is the size of ur unwanted band and wanted band? if you really want to remove the unwanted band reducing the PCR cycles number will help. but I figure in time contaminating DNA will reach the amount to cause faint band even in lower PCR cycles no. In short most likely not all contaminating template were remove.

do your pcr reagent include dutp? u can try UNG to reduce the carryover DNA from previous experiment.

and finally UV can help in reducing contaminating DNA but bear in mind UV lamp have lifespan make sure that UV lamp still good.

[pigfarmer]

You seem to have tried to change reagents, but what about your cycle?

Try increasing annealing temperature perhaps.

[lisalin]

Originally Posted by butters

actually what is the size of ur unwanted band and wanted band? if you really want to remove the unwanted band reducing the PCR cycles number will help. but I figure in time contaminating DNA will reach the amount to cause faint band even in lower PCR cycles no. In short most likely not all contaminating template were remove.

do your pcr reagent include dutp? u can try UNG to reduce the carryover DNA from previous experiment.

and finally UV can help in reducing contaminating DNA but bear in mind UV lamp have lifespan make sure that UV lamp still good.

The size of the unwanted band was 180 and 80, whereas 180 and 240 for wanted bands. And my Master Mix does not have dutp in it. Thanks for reminding me about the UV lamp!!

[lisalin]

Originally Posted by pigfarmer

You seem to have tried to change reagents, but what about your cycle?

Try increasing annealing temperature perhaps.

I ran 40 cycles and I did not make any changes though. Maybe I will recompute the annealing temperature for the chosen primers and hopefully could figure out something. Thanks a lot..!

[butters]

lisalin any new update?

[lisalin]

Originally Posted by butters

lisalin any new update?

Purchasing new reagents, and still trying to figure out what was wrong with my technique. Thanks for asking!