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My PCR is not work again


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6 replies to this topic

#1 aimmy

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Posted 19 February 2002 - 04:26 PM

I try many ways to amplify this part of gene (about 128 bp). But it's not work. First, I thought that maybe I run electrophoresis in the wrong way so I change it in TAE buffer, 50V., 2.5%agarose, 10 cm. length and it takes about 2.5 hrs.
I use phageX174/HaeIII as my marker. But I didn't have any band (the marker doesn't give all band).
So I think that instead of wasting time with optimizing PCR conditions. I'll go to hybridize my primer with my DNA samples and see it will work or not.
Is it a good way for this situation?
thanks for your comments. :D

#2 Helena Lorenzo

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Posted 20 February 2002 - 09:25 AM

Dear friend
It doesnīt matter whether you run electrophoresis with 2.5 % or 2%, you should see something in the gel for the PCR reaction if you are seeing markers. I think itīs a good idea to do blotting, the result can be even clearer.

See you


#3 scogne

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Posted 21 February 2002 - 04:02 AM

Hi,
fisrt, you have a problem of electrophoresis, you must see all the bands for the marker.Maybe you can use another one (ie 50 bp DNA ladder from promega), it would be better for 128 bp.Second try to use a fresh ethidium bromide staining solution, a 2 % routine use agarose, 100V in TBE 0.5X for the migration.
Good luck!!

#4 keren

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Posted 21 February 2002 - 11:26 AM

Hi
I have few suggestions for your problem. First: For a 50 ml agarose gel, I add 5uL of a BET solution. Be careful, BET is a charged molecule, that moves as ADN. That's why after 2.5 hours you can not see your marker of molecular weight. Second, it's not necessary to use a 2.5% agarose gel for a little fragment, or for PCR test. With 1.5%, the migration is more fast, so you have not BET migration problems. Third, did you analyzed the composition of your gene region? If it is GC rich, perhaps using DMSO 10% will render it more easy. Good luke, and if you have any question, I'll be happy to help you!

#5 5yue

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Posted 21 February 2002 - 09:18 PM

Design the primer again?
Tm......

#6 huahui

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Posted 25 February 2002 - 04:49 AM

Maybe your Taqase is bad,or your Template have been degraded.Is that so?

#7 Kenwon

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Posted 26 February 2002 - 06:15 PM

Or....... U run the sample for too long time and they ran out from the gel




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