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Only DNA ladder , No desired band in PCR


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4 replies to this topic

#1 chiang

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Posted 19 November 2009 - 04:00 AM

i need help~~ someone please~~~ i doing amplifies my LEP A19G using PCR, there's only DNA ladder apppear in my agarose gel, there is no others band i can observe other than the ladder...what happen~~
my friend with the same master mix volume, but different primer was able to get the band, is that the primer problem?
does primer expired?

#2 Maddie

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Posted 19 November 2009 - 12:04 PM

Obviously, your PCR didn't work. Can be many reasons (forgot the Taq, put the wrong concentration of one reagent, forgot the DNA, too litte or too much DNA, inhibition of some kind).
I'd just try again, paying extra attention. Can you include a positive control?
And yes, primers expire, like every reagent.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#3 DocFlow

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Posted 23 November 2009 - 12:45 AM

This is a very common problem. Try to add 1Ál MgSO4 to your reaction. when I had problems with my PCRs I started with varying annealing temperature and adding MgSO4. Usually one or both helped. also, if you have too much template this may also mess up your PCR.

#4 giftfee

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Posted 23 November 2009 - 01:55 AM

do you see at least the template bands on the gel or nothing??

#5 Prep!

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Posted 23 November 2009 - 02:55 AM

i agree with maddie
this might just be is a very trivial issue unless your positive control also does not show up anything!!!
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