John M. S. Bartlett.pdf - Bio-Nica.info

162 Abe
5. Moloney murine leukemia virus: 200 U(µL (M-MLV) reverse transcriptase (Gibco BRL).
6. Primer sequences used for detection of HBV DNA and HCV RNA by direct (RT) PCR
are listed in Table 1 (100 ng/µL).
7. Deoxy nucleotide triphosphate (dNTP) mixture: 10 mM (Pharmacia Biotech).
8. Phosphate-buffered saline (PBS): Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 ,
and 0.24 g of KH 2 PO 4 in 800 mL of distilled H 2 O. Adjust the pH to 7.4 with HCl. Add
H 2 O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for
20 min at 15 psi in. on liquid cycle. Store at room temperature.
9. RNase-free H 2 O.
10. 50-bp DNA ladder (Pharmacia Biotech).
3. Methods
3.1 Pretreatment of Serum Samples (see Note 1)
1. Serum samples (4 µL) are diluted with PBS to the final volume of 20 µL (15 dilution,
see Note 1).
2. The diluted serum samples are heated for 3 min at 95°C then cooled rapidly on ice for
3 to 5 min.
3. Five microliters of the heat-denatured diluted serum samples (= 1 µL of serum) are used
as templates for the nested (RT) PCR.
3.2. PCR Reaction
1. Set up PCR mastermixes as follows.
a. Reverse transcription and first PCR buffer (when performing DNA PCR omit RNase
Inhibitor and M-MLV Reverse Transcriptase, see step 4).
for RNA for DNA
Ingredients µL µL Final concentration
10× AmpliTaq Gold buffer 5 5 1× (1.5 mM MgCl 2 )
(containing 15 mM MgCl 2 )
10 mM dNTP mix 1 1 200 µM
Sense primer (100 ng/µL) 1 1 100 ng
Antisense primer (100 ng/µL) 1 1 100 ng
AmpliTaq Gold DNA polymerase (5 U/µL) 0.4 0.4 2 U
RNase inhibitor (40 U/µL) 0.25 None 10 U
M-MLV reverse transcriptase (200 U/µL) 0.5 None 100 U
RNase-free H 2 O 35.85 36.6
Total 45 µL 45 µL
b. Second PCR buffer
Ingredients µL Final concentration
10× AmpliTaq Gold buffer 5 1× (1.5 mM MgCl 2 )
(containing 15 mM MgCl 2 )
10 mM dNTP mix 1 200 µM
Sense primer (100 ng/µL) 1 100 ng
Antisense primer (100 ng/µL) 1 100 ng
AmpliTaq Gold DNA polymerase (5 U/µL) 0.4 2 U
RNase-free H 2 O 39.1
Total 47.5 µL