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

174 Tellier et al.
13. Thin-wall PCR tubes (Stratagene; see Note 4).
14. Robocycler 40 (Stratagene; see Note 5).
3. Methods: Long PCR
3.1. Long PCR of DNA
1. Components of the PCR are prepared in a master mix. For each reaction use the following:
10× KlenTaq PCR buffer (5 µL); 1.25 µL of dNTP mix (10 mM each) L; 1 µL of
sense primer (10-µM stock); 1 µL of antisense primer (10-µM stock); 1 µL of KlenTaq
Advantage; and 30.75 µL of ddH 2 O.
2. Aliquot 40 µL in thin-wall 0.5-mL PCR tubes and keep at room temperature. Add 10 µL
of the DNA template to the mix. Overlay with 40 µL of mineral oil.
3. Cycling parameters. We use a Robocycler 40; during each cycle of PCR we use the
following parameters (see Note 6): denaturation: 99°C × 35 s; annealing: 67°C × 30 s; and
elongation: 68°C × (optimal time for the targeted amplicon; see Note 7).
3.2. Nested Long PCR
1. We have shown that the strategy of nested PCR can be applied with success to long PCR.
It requires a slightly modified master mix, including per reaction (see Note 8): 4.5 µL
of 10× KlenTaq PCR buffer; 1.25 µL of dNTP mix (10 mM each); 1 µL of sense primer
(10 µM); 1 µL of antisense primer (10 µM); 1 µL of KlenTaq Advantage; and 36.25 µL
of ddH 2 O.
2. Aliquot 45 µL of master mix in 0.5-mL thin-wall PCR tubes and overlay with 40 µL of
mineral oil. Add 5 µL of the first-round PCR (see Notes 9 and 10).
3. Cycling parameters as in Subheading 3.1.
3.3. Long RT-PCR
3.3.1. Reverse Transcription
1. Components of the reverse transcription are prepared in a master mix. For each reaction
use (see Note 11) the following: 4 µL of 5× RT buffer; 0.5 µL of RNasin; 1 µL of DTT
(100 mM); 1 µL of dNTP mix (10 mM each); 2.5 µL of primer (10 µM; see Note 12);
and 1 µL of Superscript II.
2. Heat the 10 µL of RNA aliquot at 65°C for 2 min, and then put on ice.
3. Add 10 µL of master mix and incubate 1 h at 42°C (see Note 13).
4. Put on ice, add 1 µL of RNase H and 1 µL of RNase T1, and incubate 20 min at 37°C
(see Note 14).
5. Keep on ice until used in long PCR or keep frozen for later use.
3.3.2. Long RT-PCR
Because of buffer incompatibility (the buffer for Superscript II contains KCl), not
all of the RT reaction can be used in the PCR. We obtain good results by transferring
a small amount of the RT reaction into a long PCR mix. The remainder of the RT
reaction can then be frozen and used at a later time.
1. To amplify by long PCR the cDNA produced in the RT, we prepare a master mix as in
Subheading 3.1. but corrected for adding the template in a volume of 2 µL. For each
reaction, use the following: 5 µL of 10 × KlenTaq PCR buffer; 1.25 µL of dNTP mix
(10 mM each); 1 µL of sense primer (10-µm stock); 1 µL of antisense primer (10-µm
stock); 1 µL of KlenTaq Advantage; 38.75 µL of ddH 2 O.