The reaction mixture containing 2 [micro]g of denatured DNA; 120 [micro]mol/L each of dATP, dCTP
, and dGTP; 60 [micro]mol/L dTTP; 60 [micro]mol/L Cy5-dUTP; 50 ng/[micro]L random hexamers; and 50 U of Klenow enzyme (New England Biolabs) in its supplied 1x reaction buffer was incubated at 37[degrees]C for 1-2 h.
We used 5 [micro]L for PCR amplification in a total volume of 27 [micro]L (final concentrations) 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.8 mM Mg[Cl.sub.2]; 0.1% Triton X-100; 0.005% gelatin; 70 [micro]M each dATP, dCTP
, dGTP, and dTTP; 2.5 U AmpliTaq (Perkin-Elmer); 10 pmol upstream primer; and 10 pmol "extended" downstream primer (T7HT11V; Table 1).
Each reaction contained 50 ng of DNA, 1X PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM Mg[Cl.sub.2]; Applied Biosystems, Foster City, CA), 0.2 mM each of dATP, dCTP
, dGTP, and dTTP (Roche Diagnostics GmbH), 3 mM magnesium acetate, 1.5 U AmpliTaq DNA polymerase (Applied Biosystems), and 50 ng of each primer URA5 (5'ATGTCCTCCCAAGCCCTCGACTCCG 3') and SJ01 (5' TTAAGACCTCTGAACACCGTACTC 3').
SSR analyses were carried out in 20-[micro]L reactions with 60 ng of genomic DNA, 0.15 [micro]M of each primer, 1x Gibco-BRL PCR buffer, 2 mM Mg[Cl.sub.2], 200 [micro]M each of dGTP, dTTP, dATP and dCTP
, 0.75 U Taq Polymerase (Gibco-BRL), and 0.5x SCR dye [6% (w/v) sucrose, 100 [micro]M cresol red).
The PCR reactions for MCP-1, RANTES, CCR5, and SDF-1 were carried out in a total volume of 25 [micro]L with 5 [micro]L of extracted genomic DNA; 100 [micro]M each of dATP, dGTP, dTTP, and dCTP
; 1.5 mM Mg[Cl.sub.2; 1 U of Taq polymerase; and the two primers, forward and reverse, each at a concentration of 80 nM.
Each reaction contained 5 [micro]L of purified DNA as template in a total volume of 50 [micro]L, as well as 200 [micro]M each deoxynucleoside triphosphate (dNTP) (dATP, dCTP
, dGTP, and dTTP), 1.25 units Taq polymerase, and 0.5 [micro]M each of primer.
This mixture was incubated at 70[degrees]C for 10 min and then was mixed with 600 U of SuperScript II reverse transcriptase (Invitrogen BV/NOVEX); 30 units of RNase inhibitor; 50 mM Tris-HCl; 75 mM KCl; 3 mM Mg[Cl.sub.2]; 10 mM dithiothreitol; 0.5 mM each of dATP, dTTP, and dGTP; 0.2 mM dCTP
; and 1 mM Cy dye in a final volume of 30 [micro]L.
An Arabidopsis OLD cDNA fragment from the plasmid pF2b (Okuley et a!., 1994) was purified and labeled with [[micro]-32P] dCTP
(NEN Dupont, 3000 Ci/mmol, 10 mCi/mL) with a random-priming kit (Pharmacia Biotech, Piscataway, NJ) according to the instructions of the manufacturer.
This corresponded to 1.25 U of AmpliTaq Gold DNA Polymerase; 0.5 U of AmpErase Uracil N-Glycosylase; 200 [micro]M each of dATP, dCTP
, and dGTP; 400 [micro]M dUTP; 10x TagMan Buffer A; and 25 mM Mg[Cl.sub.2].
Amplification of bean DNA was performed in a 10-[micro]L volume containing buffer [50 mM KCl, t0 mM Tris-HCl (pH 9.0) 0.1% Triton X-100 (v/v), 1.5 mM Mg[Cl.sub.2]], 200 [micro]M each of dATP, dCTP
, dGTP, and dTTP, 0,4 [micro]M primer, 1 unit Taq DNA polymerase, and 20 ng of template DNA.
Protocols that use dCTP
require a PCR clean-up step with shrimp alkaline phosphatase to inactivate remaining nucleotides (dCTP
) that would interfere with the performance of biotin-dCTP.
Each amplification with STS213 primers consisted of 1 to 5 mg rice DNA in a mixture containing 10 mM Tris-HCl, pH 8.2; 50 mM KCl; 100 [micro] M each of dATP, TTP, dCTP
, dGTP; 2 MM Mg[Cl.sub.2]; 400 nM of each primer, STS213f and r; and 1 U Taq DNA polymerase per 25-[micro]L reaction volume.