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

PCR Fluorescence Differential Display 239
11. Thermal cycler with a ramping speed not more then 2.5°C/s. (or with programmable
ramping speed).
12. GenomyxLR DNA sequencer and GenomyxSC Fluorescent Imaging Scanner (Genomyx,
Foster City, CA).
3. Methods
3.1. RNA Isolation (see Note 1)
1. Isolate total RNA using peqGold RNAPureKit from approx 1 × 10 6 cells according to the
manufacturer’s protocol. The final volume of total RNA suspension should be 20 µL.
2. Measure OD 260 of 2 µL of RNA suspension in 80 µL of DEPC water (140 dilution). The
total yield should be approx 300 µg of total RNA.
3.2. Reverse Transcription (see Note 2)
1. Reverse transcribe 3 µg of total RNA using First-Strand cDNA synthesis Kit.
2. Place 3 µg of total RNA in a microcentrifuge tube and add RNAse-free (DEPC) water, if
necessary, to bring the suspension volume to 7.75 µL.
3. Heat the mixture for 10 min at 70°C and then place on ice immediately.
Components (concentration of stock solution) Amount Final concentration
Bulk First-Strand cDNA Reaction Mix (3×) 15 µL 1×
DTT solution (200 mM ) 11 µL 13.3 mM
Tailing primer, e.g. 5′-T 12 CA-3′ (25 µM ) 11.25 µL 2 µM
RNA suspension (3 µg) 17.75 µL 0.2 µg/µL
Final volume 15 µL
4. Mix the following components on ice according to Table 1 for one reaction.
5. Incubate for 1 h at 37°C.
6. Incubate the tube at 95°C for 2 min to inactivate reverse transcriptase.
7. Dilute the reaction mix 125. This quantity of template is sufficient for approx 180
DD-PCRs in the volume of 10 µL.
3.3. PCR (see Note 3)
1. Set up the PCR mix according to Table 2.
Components of stock solution) Amount Final concentration
cDNA (125 dilution of RT-reaction mix) 12 µL –
10× buffer 11 µL 111×
MgCl 2 (25 mM ) 10.625 µL 113 mM
random 10-mer primer (5 µM ) 10.5 µL 110.5 µM
tailing primer (25 µM ) 11 µL 112.5 µM
dNTP (1 mM of each) 12 µL 200 µM
TAMRA-dUTP (400 µM ) 10.1 µL 114 µM
Taq DNA Polymerase (5 U/µL) 10.1 µL 110.5 U
H 2 O 12.675 µL –
Final volume 10 µL
2. Prepare all the samples in duplicates to check the reproducibility of the DD-PCR banding
pattern.
3. Perform the PCR with the following settings: initial denaturing 2 min at 95°C; 40 cycles
30 s at 94°C (denaturation), 30 s at 40°C (annealing), and 30 s at 72°C (extension).
4. Use the thermal cycler with a ramping speed not more then 2.5°C/s (see Note 4).