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PCR help


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5 replies to this topic

#1 kroger

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Posted 16 March 2005 - 02:41 PM

Hi,guys:
I am repeating my PCR now. Acturally I already got my PCR product at 62C degree. But now when I repeat it,I can't get my products again. I only get the smeared band from top to the bottom of the gel. the smeared band is very bright.I don't know what's the problem of my PCR.could you give me some suggestions?
thanks a lot

#2 infrarojo

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Posted 16 March 2005 - 06:17 PM

I would trash everything and start all over. I feel your primers and dNTPs have not been handled cold all the time...
Be careful on how you hold the reagent tubes between your fingers.
DO NOT trust sterilized water from someone else. Make your own the day before.
PCR sounds like something trivial. It is not.

#3 yongyk

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Posted 16 March 2005 - 08:17 PM

Hi,
Do you quantitate your template prio PCR? When yoy have smeared band from top to the bottom of the gel most likely due to the Excessive omount of templete.

When you carry out DNA quantification be aware of you DNA purity. The ratio of A260:A280 has to be > 1.5 and you anly put < 1ug per 50ul reaction.

Try it out! Good luck!

Yongyk :P

#4 lula

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Posted 24 March 2005 - 11:34 AM

hi
after checking the DNA ,,,u can make several test tubes using the same annealing temperaure u r on
change the source of the components u r using ....in one tube change ur Taq ,,,in another one use a new amount of ur working primers
in the other change ur dNPS
sometimes one component is jus no working well

Edited by lula, 24 March 2005 - 11:35 AM.


#5 MoleculeMan

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Posted 24 March 2005 - 12:23 PM

If you think about it, the reason that you would get a smear is that you have product that is varying molecular weight. This could be due to the fact that you don't have a very clean DNA extraction before you run your PCR reaction, you could have alot of nucleases in the mix that are chewing up your template/product. It could be that the first time you ran the reaction, you got to the template before the little buggers had much of a chance to do anything. If you leave the unpurified template out of the freezer long enough it gives them time to work. Try using a good DNA extraction/purification kit. Qiuagen and promega make some column based kits that seem to work well.

#6 yongyk

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Posted 26 March 2005 - 01:50 AM

Dear kroger,

What MoleculeMan say is very true. What extraction method are you using?
Try to chech ratio of A260:A280.

Good Luck
yongyk




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