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

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2. Purify polyA + mRNA from total RNA using, for example, Poly(A)Purist kit. This original
protocol requires 2.5 to 5 µg of mRNA for the next step (but see Note 3).
3.2. cDNA Synthesis
1. Synthesize double-stranded cDNA using SUPERSCRIPT ® Choice System. For the first strand
synthesis, use 2.5 µg of the biotinylated oligo dT purchased separately, instead of that
supplied with the kit. Synthesize the second strand, proceed to the next step in this SAGE
protocol. (Do not perform EcoRI adapter addition).
2. Phenolchloroform (P/C) extract and ethanol precipitate (see Notes 5–7). Resuspend
in 20 µL of LoTE.
3.3. Cleavage of Biotinylated cDNA with Anchoring Enzyme NlaIII
to Create CATG Sticky-End
1. To 10 µL (half) of the biotinylated cDNA, add 74 µL of LoTE, 10 µL of 10× NEBuffer 4,
1 µL of 100× BSA, and 5 µL of NlaIII. Incubate at 37°C for 1 h.
2. P/C extract, ethanol precipitate, and resuspend in 20 µl LoTE.
3.4. Binding of Biotinylated cDNA to Magnetic Beads
1. Add 100 µL of streptavidin Dynabead slurry to each of two 1.5-mL microcentrifuge tubes.
Immobilize beads with Dynal magnet and remove supernatant.
2. Wash beads with 200 µL of 1× B+W buffer then remove supernatant.
3. Add 100 µL of 2× B+W buffer, 90 µL of dH 2 O, and 10 µL of cleaved biotinylated cDNA to
each of the two tubes. Incubate with gentle mixing for 15 min at room temp.
4. Immobilize beads with magnet and remove supernatant. Wash three times with 200 µL of
1× B+W buffer and once with 200 µL of LoTE. Proceed immediately to next step.
3.5. Ligating CATG Sticky-Ended Linkers to Bound cDNA
1. Linkers must be kinased and annealed in advance (see Note 4).
2. Use one of the two tubes containing washed magnetic beads for linker 1 and the other for
linker 2. Immobilize beads with magnet and remove supernatant.
3. Add 29 µL of LoTE, 5 µL of annealed linker 1 or 2, and 4 µL of 10× ligase buffer.
Resuspend the beads with gentle mixing (flick tube with finger).
4. Heat at 50°C for 2 min then allow to cool to room temp over 15 min.
5. Add 2 µL of T4 DNA Ligase. Incubate at 16°C for 2 h with intermittent gentle mixing.
6. After ligation, wash beads three times with 200 µL of 1× B+W buffer. Transfer to new tubes.
Wash once with 200 µL of 1× B+W buffer and twice with 200 µL of 1× NEBuffer 4.
3.6. Creation of cDNA Tags and Their Release
from Magnetic Beads Using Tagging Enzyme BsmFI
1. Remove buffer then add 87 µL of LoTE, 10 µL of 10× NEBuffer 4, and 1 µL of 100× BSA
to each tube. Pre-incubate at 65°C for 2 min.
2. Add 2 µL of BsmFI. Incubate at 65°C for 1 h with intermittent gentle mixing.
3. Immobilize beads with magnet. This time, collect supernatant and transfer to two new
tubes. Wash beads with 100 µL of 1× NEBuffer 4. Collect buffer and add to previous
supernatant, discarding beads.
4. P/C extract, ethanol precipitate, and resuspend in 10 µL of LoTE.