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First strand synthesis
• Purify total RNA from each ES cell line using Trizol, according to
the manufacturer’s instructions.
• Resuspend the purified RNA in 20 µl of H2O.
• Add to 10 µl of RNA, 10 pmol of LaczRT in a total volume of 15.5 µl.
• Incubate at 70°C for 10’.
• Cool on ice and add to a total volume of 24 µl:
2.5 µl 10X reaction mix
2.5 µl DTT 0.1M
1 µl dNTP 10mM
3 µl MgCl2 25mM
• Incubate at 42°C for 2 min.
• Add 1 µl SuperScript II RT and incubate at 42°C for 50 min and then
at 70°C for 15 min.
• Incubate reaction mix at 37°C for 2 min, add 1 µl RNAse mix, and
incubate for 30 min at 37°C.
Purification of the cDNA
• Using the reagents provided by the Invitrogen SNAP Column purification
system;
• Otherwise, precipitate the cDNA with 2M NH4Ac and 2V of EtOH;
• Incubate at –20°C for 30 min;
• Centrifuge at max speed for 20 min;
• Wash the pellet twice with 70% EtOH and centrifuge at max speed for
2 min.
• Resuspend the pellet with 50 µl of H2O.
TdC tailing of cDNA
To 10µl of first strand of cDNA, add:
5µl 5 x Tailing Buffer
2.5µl 2 mM dCTP
2.5µl H20.
• Incubate at 70 72°C for 5’.
• Cool on ice for 1 min exactly.
• Add the following reagents
• 0.2µl 5x Tailing Buffer
• 0.3µl TdT
• 4.5µl H20
• Incubate at 37°C for 10’
• Incubate at 72°C for 10’
• Cool down to 20°C.
PCR of dC-tailed cDNA
To 5µl of dC-tailed cDNA, add:
• 5µl of 10x PCR buffer
• 3 µl of 25mM MgCl2
• 1 µl of 10mM dNTP
• 2 µl of 10 µM of L232
• 2 µl of 10 µM of AAP
• 0.5µl of Taq DNA polymerase
• H2O to a final volume of 50µl
Perform 30 to 35 cycles of PCR with an annealing temperature of 55°C.
Nested amplification
Dilute a 5 µl aliquot of the primary PCR in 495 µl TE buffer.
To 5µl of diluted primary PCR product, add:
• 5µl of 10x PCR buffer
• 3 µl of 25mM MgCl2
• 1 µl of 10mM dNTP
• 2 µl of 10 µM of LacZ
• 2 µl of 10 µM of UAP
• 0.5µl of Taq DNA polymerase
• H2O to a final volume of 50µl
Perform 30 to 35 cycles of PCR with an annealing temperature of 58°C.
Analyze 5 to 20 µl of the amplified sample, using agarose gel electrophoresis,
ethidium bromide staining and the appropriate molecular size standard.
The sharp band can direst sequenced.
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