carrier/blocking nucleic acids for ISH

Dunowska, Magda via methods%40net.bio.net (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? 
Thanks, 
Magda

-----Original Message-----
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:
>
>hello all 
>I am trying to pcr out a sequence  with pfu. This
>> has worked before perfectly   with taq, but now I get repeatedly emplty
> lanes.
>> 
>>  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 
lose. 

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 
>product?

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. 

DK
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