I understand part of this didn't come through or was not completely
edited from the WordPerfect version so I am sending it again.
>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
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 =3D<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.
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
Table 1 Reaction Mixtures for PCR Labeling
Final For DNA amplicons
<500 bp >500 bp
Use Use of Solution
20 =B5l 8.2 =B5l 3.2 =B5l 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
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=A7 a-32P-dATP (3000 Ci/mmol)
*- Idaho Buffer 1x concentration =3D 50 mM Tris, pH 8.3; 250 ug/ml
BSA; 1 mM MgCl2; 0.5% ficoll; 1 mM tartrazine.
=A7- 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
"They named it GOLF because all the other four letter
words were taken." Lee Trevino
|| Douglas Rhoads || =
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