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

200 McDonagh
2.2. Basic PCR
1. dNTPs (2 mM, 10× stock; Pharmacia; see Note 2).
2. Taq or Pfu DNA polymerase and 10× buffer (Sigma, Promega; see Note 3).
3. Forward and reverse primers (2 µM; 10× stock).
4. Mineral oil (Sigma) or wax beads (Perkin–Elmer, Warrington, UK).
5. Thermocycler (Hybaid, Teddington, UK).
2.3. PCR to Create Mutated Competitor Fragments
(e.g., Containing a Novel RE Site)
1. Forward and reverse primers used in conventional PCR (2 µM).
2. Internal forward and reverse primers containing bases to create novel RE site (2 µM).
3. DNA purification system (Geneclean, Bio 101, US).
4. PCR cloning system (TA cloning kit; Promega or Invitrogen; Leek, The Netherlands).
5. RE enzyme (to digest mutated product) or method of differentiating between wild-type
and mutated product, such as a probe.
3. Methods
3.1. Reverse Transcription
1. Mix together the following reagents in an ice bath:
7.5 µL of RNAse free water
3 µL of DMSO
100 ng of antisense virus-specific primer (in 1 µL)
2 µL × 10 RT buffer
3 µL of 1 mM dNTPs
0.5 µL of RNAsin (20–40 units/µL)
5 µL of RNA, prepared by extraction method
1 µL of RT
2. Incubate for 30–60 min at 42°C followed by 5 min at 95°C and 5 min at 4°C.
3. Pulse spin tubes and add 5 µL to PCR reaction or store at –20°C until required (see
Note 4).
3.2. PCR
3.2.1. Basic Reaction
1. Make up a master mix containing 5 µL of buffer (×10); 5 µL of dNTP (2 mM, final
concentration 0.2 mM); 5 µL of each primer (2 µM, final concentration 0.2 µM); and 1 U
DNA polymerase (see Notes 2 and 3).
2. Overlay with oil if not using a thermocycler with a heated lid, then add 5 µL of prepared
cDNA through the oil.
3. A typical cycling protocol consists of denaturation at 95°C for 30 s, annealing at 5°C below
the primer melting point for 30 s, and extension at 68 to 74°C for 45 s (see Note 5).
4. Carry out as few cycles as necessary because the reaction is only linear over a short range
and this may distort the results.
5. To further increase sensitivity, it is beneficial to use nested PCR. As a general rule, the
primary round should consist of 20 to 25 cycles and the secondary round up to 20 cycles. It
may also be necessary to add diluted primary product (1/10) to the secondary round.
3.2.2. Limiting Dilution Method
A limiting dilution of cDNA is performed in 10-fold steps, and this is then added
to the PCR. The sensitivity of the assay must be known, and the amount of cDNA in