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

cDNA Libraries from Few Cells 507
3.5. Ligation of the ss-cDNAs to the Modified Oligonucleotide
(see Note 4)
1. Remove primer before the PCR amplification step.
3.6. PCR Amplification (see Notes 5 and 6)
The general conditions used for both PCR amplifications are: 50 µL of reaction
volume, hot start, and touchdown PCR.
1. First PCR: Use A5′_1 and A3′_1 and half of the purified ligation mixture.
2. Second PCR: Use A5′_2 and A3′_2 and one tenth of the purified first PCR.
3. For both PCRs:
a. Use as final concentration 200 µM of dNTPs, 0.8 µM of each primer, and 1.5 mM
of MgCl 2 .
b. Hot start: Add 0.5 µL of Taq DNA polymerase (5 U/µL) below the mineral oil when
the reaction mixture reaches 80°C.
c. Perform the following touch down PCR cycles: denaturation 93°C for 3 min; two cycles
of 94°C, 30 s/70°C, 45 s/72°C, 1.5 min; two cycles of 94°C, 30 s/69°C, 45 s/72°C,
1.5 min; two cycles of 94°C, 30 s/68°C, 45 s/72°C, 1.5 min; two cycles of 94°C,
30 s/67°C, 45 s/72°C, 1.5 min; two cycles of 94°C, 30 s/66°C, 45 s/72°C, 1.5 min;
25 cycles of 94°C, 30 s/65°C, 45 s/72°C, 1.5 min; and cool down to 4°C.
d. Remove primer after each PCR.
After the second nested PCR the amount of ds-cDNA is about 2 to 5 µg.
3.7. Direct Sceening of the PCR Library
with Biotinylated Oligonucleotides
3.7.1. Hybridization with the Biotinylated Oligonucleotide (see Note 7)
1. Use 500 ng of purified ds-cDNA. The volume should not exceed 8 µL.
2. Add 2 µL of the 100 ng/µL biotinylated oligonucleotide.
3. Adjust volume to 10 µL.
4. Heat denature for 5 min at 95°C.
5. Immediately add 100 µL of hybridization buffer: 5× SSPE, 5× Denhardt’s solution,
1% SDS.
6. Incubate overnight at 42°C.
3.7.2. Separation of Probe-cDNA Hybrids
After the hybridization reaction, probe-cDNA hybrids are separated from unhybridized
DNA using Streptavidin-coated magnetic beads.
1. Prehybridize Dynabeads with salmon sperm DNA by washing 20 µL of 10 mg/mL
Dynabeads twice with 50 µL of hybridization buffer containing 250 µg/mL salmon sperm
DNA. Incubate for 2 h at room temperature on a rotating wheel.
2. Mix the Dynabeads with the probe-cDNA solution. Incubate 15 to 30 min at room
temperature on a rotating wheel.
3. The hybrids captured by the beads are washed twice with 1× SSC, 1% SDS, then twice
with 0.1× SSC, 1% SDS. Washes are performed at 42°C for 20 min each.