PCR reaction

[charmaine]

Hey guys,

was wondering about the problems involved in PCR reactions on the agarose gel.

Was wondering what the problems were when you get smearing on the gel.

 

I have it listed as a problem with annealing temperature or mgcl concentration.

 

Are there any other reasons as to why you would get smearing?

 

thankyou

[ecoli]

You question is very confusing.You don't run a PCR reaction in an agarose gel, you know. That's what it sounds like your doing.

[charmaine]

Sorry. I'm very new to all of this.

What I am hoping to find out arethe problems that cause smearing of the bands on the agarose gel.

eck.

[ecoli]

Sorry. I'm very new to all of this.

What I am hoping to find out arethe problems that cause smearing of the bands on the agarose gel.

eck.

 

Ohhh... I apologize, I didn't understand your question. The answer is yes. If the genes don't amplify properly then the banding could be smeared, as if you just loaded DNA in the sample instead of PCR product.

 

You should run the PCr reaction again, varying the annealing temperatures, etc. That might work for you.

 

Also, look up some online resources, a lot of that sort of info is already documented.

[Hailstorm]

http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html

 

Good luck.

 

Also, what's the GC content you're attempting to clone?

 

Are your primers good?

[hyebeh]

man... if only i had access to these supplies. damn my high school. lol

[ecoli]

man... if only i had access to these supplies. damn my high school. lol

 

I know what you mean... my school has absolutely no equipment for even slightly advanced labs. Our electrophoresis lab consisted of paper, cardboard and glue.

[hyebeh]

haha... we just read the labs and the teachers makes up fake results for us. the only labs we do are the labs that cost little or no money (e.g. plant and enzyme labs)

[Guest BizBomb]

my school is pretty rich so we got to do quite a few pretty good experiments, but never PCR. We did a pretty good electrophoresis lab, and we got real results too. It took a really long time though.

[ecoli]

Our enzyme lab consited of some liver from the super market.