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

102 Stirling
Table 1
Thermal Cycler Program for Subcycling PCR
Step Temp/command Time (mm:ss)/repetitions
11 95 2:00
12 94 00:10
13 63 00:05
14 68 5:00
15 Go to step 3 4 items
16 Go to step 2 15 times
17 94 00:10
18 63 00:10
19 68 6:00 (+ 10s per cycle)
10 Go to step 8 4 times
11 Go to step 7 15 times
12 68 30:00
13 End
6. Oligonucleotides: INT22-P (5′-GCC CTG CCT GTC CAT TAC ACT GAT GAC ATT ATG
CTG AC-3′); INT22-Q (5′-GGC CCT ACA ACC ATT CTG CCT TTC ACT TTC AGT
GCA ATA-3′); INT22-A (5′-CAC AAG GGG GAA GAG TGT GAG GGT GTG GGA
TAA GAA-3′); and INT22-B (5′-CCC CAA ACT ATA ACC AGC ACC TTG AAC TTC
CCC TCT CAT A-3′). The oligonucleotides are diluted to 100 pmol/µL for storage.
Subaliquot to minimize freeze-thaw cycles and store at –20°C.
7. PCR Mastermix 1. For each reaction to be run, the following are included: 10× buffer 2
(2.5 µmL); TAQ mix (0.94 µL); and water (5.69 µL).
8. PCR Mastermix 2. For each reaction to be run, the following are included: 10 mM aATP
(1.25 µL); 10 mM aTTP (1.25 µL); 10 mM aACP (1.25 µL); 10 mM aGTP (0.625 µL);
10 mM deaza aGTP (0.625 µL); P/Q primer mix (5 µL); A (or B) primer mix (5 µL);
100% DMSO (1.875 µL).
3. Procedure
1. Remove reagents from freezer and allow to thaw fully then mix thoroughly and briefly
centrifuge before pipetting. Reagents to be thawed: buffer 2 from Expand Long Template
PCR Kit, dNTPs, and deaza dGTP.
2. Label 0.2 mL of flat-cap PCR tubes with the DNA number and primer grouping, if
applicable (e.g., APQ).
3. Using sterile pipet tips, pipet 0.5 µL of each DNA sample into the appropriately labeled
tube. Ensure that the DNA is pipetted directly into the bottom the tube. If concentration of
DNA is very low, 1 µL of DNA should be added. Place tubes on ice.
4. Prepare 1 in 50 dilution of A oligo, 1 in 50 dilution of B oligo, and 1 in 25 dilution of
P and Q oligo pairing. The number of samples being tested will determine the actual
quantities required.
5. Prepare PCR mastermix 1 and 2 for appropriate number of tests: Place both mastermixes
on ice!
6. For each reaction, aliquot 14.9 µL of Master Mix 2 into the labeled PCR tubes (which
already contain the DNA template). Keep on ice.
7. Immediately before amplification, add 9.2 µL of Master Mix 1. Spin briefly and proceed
to PCR step immediately.