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

cDNA Libraries from Few Cells 503
Fig. 3. Alternative oligonucleotides used for subtractive cDNA library construction. For
the B oligonucleotides the same modifications (5′ phosphate and 3′ amino) as the A primers
have to be used.
2. Materials
2.1. Oligonucleotides
1. PCR library primers: For the 5′ end of the ss-cDNA, use RA3′ NV and the related
oligonucleotides (A3_1,2,3). For the 3′ end use A5′NV, A5′_1,2,3. All oligonucleotides
must bear 5′ and 3′ hydroxyl group. Only A5′NV must have a phosphate group to its
5′ end to allow ligation to the 3′ end of the ss-cDNA. To avoid selfligation of A5′ NV, its
3′ end must be protected with an amino group. Those modifications are performed by any
oligonucleotide suppliers. Apply the same rules when using B5′ NV and RB3′ NV. Note
that only two related oligonucleotides are available for both ends.
2. Biotinylated screening primers: Ask your oligonucleotide supplier for 5′ biotinylated
primers with a seven carbon spacer. Do not use an 11 carbon spacer because this will
dramatically decrease the capture yield. If this primer is designed to hybridize in the middle
of a molecule, add 4 to 6 random nucleotides at the 5′ end to facilitate the interaction with
the streptavidin magnetic beads.
2.2. RNA Extraction (see Note 1)
1. Starting material: Fresh pelleted cells, store at –80°C on collection. Fresh pieces of organs,
or tissue frozen in liquid nitrogen and stored at –80°C.
2. PolytronR TP1200 (if organs are used).
3. RNAZol reagent (BIOPROBE).
4. DT40 (Pharmacia, [Piscataway, NJ]; #17-0270-01) Prepare a 5 mg/mL stock solution in
ddH 2 O. Aliquot and store at –20°C.
5. CHCl 3 .
6. Isopropanol.
7. 100% Ethanol.
8. 70% Ethanol.