(Fwd) Correction

Douglas Rhoads drhoads at MERCURY.UARK.EDU
Wed Oct 23 17:18:55 EST 1996


Does anyone on this group know what causes a perfectly normal looking 
ASCII document to become laden with these '3D' characters and the 
like.  I am editing this one more time.  If it doesn't look good this 
time then we'll have to forget it.  Sorry for garbaging the group 
with this.

------- Forwarded Message Follows -------
To:            yeast at net.bio.net
From:          drhoads at mercury.uark.edu ("Douglas Rhoads")
Subject:       Correction
Date:          23 Oct 1996 12:36:59 -0700

I understand part of this didn't come through or was not completely 
edited from the WordPerfect version so I am sending it again.


>Hi netters,
>I'm looking for a protocol to label a probe by PCR with alpha P32dATP.
>Does somebody have such a protocol? Thanks for your help.
>                      David Humair




PCR Labeling

ref: Dana Heiny and Doug Rhoads  modification of  Schowalter and Sommer 
1989, Analytical Biochemistry:177:90-94.

For the production of very hot probes.  Probably only efficient for
amplicons <1100 bp.  Efficient PCR labeling requires low
concentrations of unlabeled deoxynucleoside triphosphates (dNTPs)
relative to standard PCR.  For longer amplicons (>500 bp) the
limiting amount of  a-32P-dATP causes accumulation of short products. 
For longer inserts up to around 1100 bp, doubling the dATP
concentration usu ally allows production of full length amplification
products.  Amplification is from =<1 ng of a target PCR fragment or
insert-containing-plasmid.  The size and source of the DNA insert to
amplify (am plicon), as well as the buffer formulation can affect the
final percent incorporation.  Since incomplete extension of the
primers results in undesirable small products (30-100 bp), total TCA
precipit able counts are poor indicators of the quality of the probe
synthesized.  Therefore, for new amplicons or those of larger size
you should determine an approximate size for the products by
resolving a small aliquot (1/500th of final product) on a small TEB
denaturing polyacrylamide gel.  Incorporations generally range from
50- 90% and produce up to 20 ng of probe at 4-6 x 10^9 cpm/ug.  The
major a dvantage of this method over nick-translation or random
priming is that final probe is not contaminated with cold template
(i.e., specific activity 6-10x higher).  One disadvantage of this
method is the size limit of the amplified region (amplicon).

The following protocol represents optimal conditions for samples
sealed in glass capillary tubes and cycled in a Idaho Technology
1605 Air Thermo-Cycler.


PCR Parameters:     
90oC 30 sec 
90oC 15 sec    \
45oC 15 sec    >   25x 
72oC 90 sec    /    Ramp set to S1 
72oC 3 min

Anneal temperature should be adjusted for the particular primers in
use according to what normally works for the primers being used.
Times for anneal and extension may also be adjusted to fit
particular PCR methods and machines other than the Idaho Aircycler.
At end of cycling, add 180 ul TE and 5 ug carrier yeast RNA.  Purify
over G-50 spin column.  Quantitate by TCA precipitation and possibly
test on denaturing mini gel.  Should be used within one day as a
hybridization probe. 

Table 1 Reaction Mixtures for PCR Labeling

Final	For DNA amplicons
           <500 bp 	>500 bp 
            Use    Use   of Solution
20 ul	   8.2 ul  3.2 ul   H2O
1x Buffer  2 ul	   2 ul	    10x Idaho Buffer*
2.5 uM	   1 ul	   1 ul	    50 uM dCTP, dGTP, dTTP
250 nM	   1 ul	   1 ul	    5 uM forward primer
250 nM	   1 ul	   1 ul	    5 uM reverse primer
50 pg/ul   1 ul	   1 ul	    1 ng/ul plasmid
                             containing amplicon
0.125 U/ul 0.8 ul  0.8 ul   3 Units/ul Taq DNA
                             Polymerase (5 Units/ul
                             use 0.5 ul)
0.825 uM   5 ul	    --	    a-32P-dATP (3000 Ci/mmol)
1.65  uM    --	    10 ul**  a-32P-dATP (3000 Ci/mmol)

*- Idaho  Buffer 1x concentration= 50 mM Tris, pH 8.3; 250 ug/ml
 BSA; 1 mM MgCl2; 0.5% ficoll; 1 mM tartrazine. 

**- the amount of total radioactivity may be reduced to 5 ul by
substitution (supplementation) with cold dATP (i.e., 5 ul of 3.3 uM
dATP). 


||                  Douglas Rhoads                        ||
||drhoads at mercury.uark.edu || Dept. of Biological Sciences||
||drhoads at comp.uark.edu    || 601 Science Engineering     ||
||501-575-3251             || University of Arkansas      ||
||FAX 501-575-4010         || Fayetteville, AR 72701      ||
||      http://comp.uark.edu/~bioinfo/bisc.html           ||
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