carrier/blocking nucleic acids for ISH
(by M.Dunowska from massey.ac.nz)
Wed Jan 13 17:19:00 EST 2010
Hi everybody, I have a quick question related to carrier/blocking nucleic acid used as part of hybridization buffer for ISH. Some protocols use salmon sperm DNA, some tRNA and some both. We are trying to detect an RNA virus with a DNA DIG labelled probe. Is there any advantage to use one versus another versus both?
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of DK
Sent: Tuesday, 11 August 2009 12:33 p.m.
To: methods from magpie.bio.indiana.edu
Subject: Re: pfu pcr
In article <mailman.762.1249923591.21502.methods from net.bio.net>, malihe keramati <mlh_keramati from yahoo.com> wrote:
>I am trying to pcr out a sequence with pfu. This
>> has worked before perfectly with taq, but now I get repeatedly emplty
>> I have tried to work with longer extension times as well
>> as annealing times,gradint pcr and increased enzyme, dnttp's ,
>> primers and mgso4 - but no success - also I digested the
>> genomic dna (bacterial ) and checked them for pcr . I keep getting
>> a gel with not the faintest track of a band.
>-Pfu and its buffer and dntp mix are functional because I check them by
> positive control and get a sharp band)).
>- I have checked these temperature :40,45,50,53,56,58,60.for pfu .
>taq pol pcr has sharp band in range of 52 to 62 C, lower temperature for taq
> has nonspecific band .
>the mg conc increased to 3mM and dNTP 0.25 mM of each and pfu conc is 1.25
> u/25 microlit .
>- the segment is about 1300 bp and extension time was set for 3 min in pfu
> reaction . However in all of experiences there is no pcr product, no band
> just primer dimmer,
In about 10 years, I had only one case like this. The solution was:
extend at 65C instead of 72C. The gene in question had stretches
of ridiculously high AT (over 90%). None of the conventional additives
worked but changing extension temp did. Try it, you don't have a lot to
In general, I wholeheartedly agree with the recommendation of
PfuFusion. In terms of fidelity and processiveness, it has so far
no equal (that I know of).
To Peter Ellis:
>What can you do with a pfu product that you can't do with a Taq
First to come to mind:
1. You can use it for efficient blunt end cloning without extra steps.
2. You can save money by not sequencing insane number of clones
if your insert is ~ 5 kbp. In fact, if you can take P=0.05 (not that I'd
recommend it), you can just skip sequencing altogether.
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