Biology Reference
In-Depth Information
5. Add 7
m
l of the first-strand Master Mix to each denatured sample from step 1.3. Keep on
ice.
6. Mix well by pipetting 10 times.
7. Quick-spin to force contents to bottom of tube.
8. Add 3
m
l of AffinityScript HC Reverse Transcriptase to each reaction (final volume of
l).
9. Incubate at room temperature for 1 h.
10. Incubate in a thermocycler at 42
C for 1 h.
11. Add 10 ml of 1 M NaOH to each reaction and incubate at 70
C for 10 min to hydrolyze
RNA.
12. Cool to room temperature slowly; do not cool on ice.
13. Centrifuge the tube briefly to collect contents.
14. Add 10 ml of 1 M HCl to neutralize the solution.
Step 2. cDNA Purification
1. Quick-spin and add 4 ml of ice-cold 7.5 M ammonium acetate and 1 ml 20 mg/ml
glycogen to each reaction from step 1.14.
2. Add 100 ml of ice-cold 100% ethanol. Mix well by pipetting and transfer to a 1.5-ml
tube.
3. Incubate at 20
C overnight. The reaction can be stored at this point for several
days or up to 2 months.
4. Centrifuge the reaction from step 2.3 at 13,000-14,000
g
for 15 min at 4 C.
Carefully decant supernatant.
5. Wash with 0.5 ml ice-cold 70% ethanol and centrifuge at 13,000-14,000
g
for
15 min at 4
C. Carefully decant supernatant and allow to air dry or dry in vacuum
dryer. Do NOT overdry.
Step 3. NHS-Ester Dye Coupling Reaction
1. Resuspend the cDNA pellet in 5
20
m
m
lof2
coupling buffer. If a precipitate can be
coupling buffer, incubate the buffer at room temperature or 37
Cto
re-solubilize the precipitate before use.
2. The first time a tube of dye is used, resuspend in 45
seen in the 2
l of the high-purity DMSO
provided in the kit. Vortex gently to ensure the pellet is completely solubilized.
Prepare single use aliquots of the unused dye, which can be stored at
m
20
C in the
dark for several months.
3. Add 5 ml of Cy3 dye to the cDNA. If the dye was stored at 20
C prior to use, allow
the dye to reach room temperature before opening the container.
4. Mix by gently pipetting up and down.
5. Incubate for 30 min at room temperature in the dark.
Step 4. Dye-Coupled cDNA Purification
1. Add 90 ml of RNase/DNase-free water to the 10 ml labelled cDNA from step 3.5.
2. Combine 100 ml of DNA-binding solution and 100 ml of 70% (v/v) ethanol. Mix
well by vortexing. Make sure that the two solutions are well mixed prior to use.
3. Add the 200 ml of DNA-binding solution/ethanol mixture to the labelled cDNA from
step 4.1 and mix by vortexing.
4. Using a pipette, transfer the mixture directly on the filter of a microspin cup that is
seated in a 2-ml receptacle tube. (Exercise caution to avoid damaging the fibre
matrix with the pipette tip.) Snap the cap of the 2-ml receptacle tube onto the top
of the microspin cup. Note: To ensure proper sample flow, use the receptacle tube
that is provided with the microspin cups.
5. Centrifuge the tube in a microcentrifuge at maximum speed for 30 s. Note: The
labelled cDNA is retained in the fibre matrix of the microspin cup.
Continued
