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Is necessary to use pfu polymearse for PCR amplification of pre-miRNA for clonin


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#1 piggle

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Posted 07 September 2009 - 07:46 PM

Hi, all
I wonder if I'm necessary to use pfu polymerase for PCR of pre-miRNA. ;)
I think that several taq polymerases have no proof reading activity. So, pre-miRNA which i want to clone may have incorrect sequence.
It have impact to secondary structure or pre-miRNA or miRNA.

Anybody have suggesion for me.
Thank for help
Piggle. :)

#2 yushulai

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Posted 08 September 2009 - 12:26 AM

It's always dependent on the length of your miRNA.
pfu is ok. But if your fragment is less than 1kb, you can use taq. Pick several clonings for sequencing (usually 3-5), then chose the right one.
In our lab, we use taq:pfu=30:1 (Sorry, I'm not sure. You can try).
Good luck.

#3 miRNA man

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Posted 08 September 2009 - 05:33 AM

As yushulai said, as long as you sequence to make sure no errors it's ok. Although for what it's worth, I've not had good results with pfu in my hands, never got much amplification, but I get good results using Herculase polymerase from stratagene (I have no affiliation with them!). Is has proof reading and good processing to get good amplification (and isn't too expensive, either).

#4 vetticus3

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Posted 08 September 2009 - 07:13 PM

pfu is a bit finiky (i have to use DMSO for it to work well)... but there are other polymerases that are proof readers. often companies will give you free samples, especially if they are under the impression that more will be purchased. :wacko:

V

#5 piggle

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Posted 08 September 2009 - 11:10 PM

thank alot. :lol:
I decide to use taq pol. of BIOTOOL
and select cloning colony around 5 for sequence and select the righ one for transfect.
:)
piggle




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