The detail of ingredients used is: Genomic DNA template 50 ng; dNTP
(2.5 mmol/L, TakaRa) 2 uL; Both primers (0.4 mol/L) 2 uL; DNA Taq polymerase (1.5U, TaKaRa) 0.5 uL and 10 x PCR buffer 5 uL.
For the second round, 3 [micro]l of the first PCR product was added to the PCR mixture (final volume of 50 [micro]l) containing 3 [micro]l of both inner primers, 10 [micro]l 5xPCR buffer (including 1.5 mmol/L MgCl [sub]2), 1 [micro]l 10 mmol/L dNTPs
(2.5 mmol/L of each dNTP
), 1.5 U PromegaGoTaq polymerase, and water to 50 [micro]l.
The reaction mixture consisted of l.0[micro]g of fungal template DNA, 50pmol of each primer, Mg[Cl.sub.2]-free reaction buffer, 2.0mM Mg[Cl.sub.2] , 0.5U of Taq polymerase and 0.2mM of each dNTP
. PCR was carried out under the following conditions: one cycle at 94[degrees]C for 5min; 30 cycles at 94[degrees]C for 3min, at 50[degrees]C for l.25min and at 72[degrees]C for l.40min; and at 72[degrees]C for 10min in a final extension.
A 25-[micro]L reaction mixture, which contained sterile distilled water (9.8 [micro]L), forward primer (0.5 [micro]L), reverse primer (0.5 [micro]L), Mg[Cl.sub.2] (1.5 [micro]L), deoxynucleotide triphosphate (dNTP
) mixture (2.5 [micro]L), genomic DNA (4.0 mL), dimethyl sulfoxide (DMSO) (2.5 [micro]L), Taq DNA (0.2 [micro]L), and buffer (2.5 [micro]L), was prepared for each sample.
After varying cycle numbers, the amount of polymerase, and the amount of deoxyribonucleotide triphosphates (dNTPs
), it was found that restricting PCR amplification was best done by using a dNTP
concentration of 3-12 [macro]mol/L.
EGCG, EA, and RA dose-dependently inhibited the growth of human HL-60 promyelocytic leukemia cells, exerted strong free radical scavenging potential, and significantly imbalanced nuclear deoxyribonucleoside triphosphate (dNTP
) concentrations without distinctly affecting the protein levels of RR subunits (R1, R2, p53R2).
The PCR beads contain 2.25 units of puReTaq DNA polymerase, 100 [micro]M of each dNTP
in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, and 1.5 mM Mg[Cl.sub.2] when used in a total volume of 25 pl.
5 unit Taq DNA polymerase and 0.2 mM of each dNTP
in a solution of 10 mM TrisHC1 50mM KCl and 1.2mM MgCl2 (Promega USA).
The reaction composition was: 1 [micro]l of DNA sample (from stock, or dilutions 1:10, 1:100, 1:200 or 1:400; diluted DNA samples were used to overcome inhibition effects of Taq DNA polymerase by possible contaminants carried over from the insect lysis), 1 U of Taq DNA polymerase, 1 [micro]M of each primer, 2 mM Mg[Cl.sub.2], and 200 [micro]M of dNTP
. The polymerase chain reaction (PCR) cycle was: initial denaturation at 92[degrees]C/3 min, followed by 30 cycles of 92[degrees]C/30 sec, 50[degrees]C/1 min, and 70[degrees]C/1 min.
Each 25 [micro]L PCR mixture contained 1 [micro]L (1:10 dilution) community DNA (10 ng-20 ng), 2.5 [micro]L PCR buffer (1X), 1 [micro]L of each deoxyribonucleoside triphosphate (dNTP
) (100 mM), 1 [micro]L of forward and reverse primers (0.5 [micro]M), and 0.5 [micro]L Taq polymerase (3U) (Fermentas).
The reaction contained 25ng/[micro]L oligo [(dT).sub.12-18], 500 [micro]M each dNTP
, 75 mM KCl, 3mM Mg[Cl.sub.2], 10 mM DTT, and 200 units of reverse transcriptase (SuperScript II RNase H-reverse transcriptase, Invitrogen, USA) in 50 mM Tris-HCl buffer (pH 8.3) in a final volume of 10 [micro]L.