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Non especific band in PCR...

PCR ANEALING TROUBLESHOOTING

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#1 lootches

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Posted 26 December 2014 - 06:21 AM

Hello guys;

I have a question about my PCR reaction,...

my original band is 78 bp( an aptamer)

when I run it ,instread of 78 bp band I have 120 bp I try to change Mg concentration and Tm but it couln't help :(

I test these one;

 

Mg; 0.6-0.7-0.8-0.5

Tm: 62,60,64

I aslo change my Primer concentartion but it doesn't help too :(

(my especific band is 78, 120 bp is nonespecific one)

 

 

please help me or say some sugestion

thank you



#2 Tabaluga

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Posted 26 December 2014 - 07:40 AM

Have the primers been validated before ? If in doubt, you could design a new set of primers (they are relatively cheap) and give it a try.


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Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 Tick

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Posted 16 January 2015 - 12:42 PM

I Agree with Tabaluga. What is the original tm of your primers? You could always try to decrease your tm. You will keek the 120 bp band, but it could be possible that your expected amplicon will appear. Hope this helps (:







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