Technique / Molecular Biology / PCR / Standard PCR


PCR contamination: amplification in negative control



PCR contamination: amplification in negative control

PCR product band is observed in agarose gel electrophoresis from a negative control.

Solution:
1. Work in a pre-PCR workspace ( i.e. PCR station with UV light) to avoid contamination from previously amplified DNA
2. Put on a fresh pair of gloves when begining work PCRs. Change gloves frequently.
3. Prepare your own sets of reagents and store them in small aliquots. When preparing these reagents, use RNase? DNase free and steriled tubes.
4. It is best to add all components of the reaction to the microfuge tube before adding the template DNA.
5. Whenever possible, include a positive control as well as a negative control that contains all the components of the PCR except the template DNA.

Last update 28-Feb-2002, Rating of 6 votes.

Post your message in Standard PCR forum

Write your comment

By Montse on 10-Feb-2009
No sé el tamaño de la banda de tu cotnaminación pero es posible que sean dímeros de primers. En las muestras no se ve porque la polimerasa se "entretiene" con lo que tiene que amplificar pero en el control negativo, como el único DNA que hay son los primers, se dedica a hacer PCR con ellos.
Espero haberte sido de ayuda.
Rating:

Reply
By JC on 01-Dec-2008
yo tengo este problema de contaminacion del control negativo, ya he cambiado todos los reactivos y tomo todas las precauciones debidas, (UV, guantes, pequeñas alicuotas de reactivos, etc).

Lo extraño de mi contaminacion es que la banda observada en el control negativo es de un peso menor al de la muestra problema (control positivo), por lo cual puedo deducir que la contaminacin no proviene de la muestra. Otra cosa tambien extraña es que las muestras no se ven contaminadas por esta banda de menor tamaño, teoricamente si la contaminacion esta en el blanco tambien deberia de estar en las muestras, en otras palabras se deberian de ver 2 bandas en las muestras, pero no es asi.

Me vendria muy bien algunos comentarios o sugerencias.
Rating:

Reply
By henry on 06-Mar-2008
i am getting Band in negative control,i hvae changed all reagents, still i am getting band. any idea..
henry
Rating:

Reply
By vandana on 12-Jul-2007
I'm trying with different conc in cocktail preparation but not getting amplification after PCR is over. where could be the mistake?
Rating:

Reply
By wei on 31-Oct-2005
Try using it with the advantage CDNA pol from Clontech. It worked for me. You may have to change the PCR parameters a bit though, depending on your system.
Rating:

Reply
By kelp on 01-Sep-2005
I have used the kit from Clontech without the advantage genomic polymerase mix along with the kit, And had got a satisfied results. So, I do not think it is necessary.
Rating:

Reply
By suki on 17-Feb-2004
try using different blunt ended enzymes to digest your DNA. Perhaps the enzymes in the kit do not cut close to your gene primer site. Also make sure that your DNA is clean and properly digested. Good luck!!
Rating:

Reply
By Hammou OUBRAHIM on 28-Feb-2002
Hi every one,

I am currently using genome walker kit from Clontech and advanced genomic PCR kit. I am not sucessfull to get any PCR products. Your suggestions are welcome.

Thanks in advance

Hammou
Rating:

Reply


Please Login or Register to Post