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

cDNA Libraries from Few Cells 509
4. Notes
1. All the material used in this manipulation must be very clean and at least sterilized. Wear
gloves throughout the manipulation to avoid RNase contamination. The same precautions
should be followed in the synthesis of the ss-cDNA. These precautions represent the lower
level of protection against RNase and it is advisable to read ref. 6 carefully. Because
PCR has to be performed later on this material, use anticontamination tips and aliquot
every solution.
2. During this manipulation, prepare controls that will be used in the ligation and PCR
experiments. Prepare samples without AMV RTase and samples without RNA. This will
lead to three different controls.
3. We have successfully used as little as 10 ng of polyA + RNA. Do not exceed 1 µg of
total RNA.
4. Prepare nonligated samples composed of the same mixture as described in Subheading 3.5.
without the T4 RNA ligase. Include each control of the cDNA synthesis.
5. After removal of the primers, perform PCR amplification. Do not forget to include a PCR
control without DNA for both amplifications.
6. To analyze each PCR amplification, load 5 µL of (one tenth) of the PCR product on an
agarose gel. After ethidium bromide staining, a signal could be observed after the first
PCR but it is generally obtained after the second nested PCR.
7. We have used degenerated primers to screen the PCR library. The amount of primer and
Dynabeads should be at least five times above the amount described in Subheading 3.7.
8. We have developped a direct PCR analysis to determine the sizes of inserted fragments
and to rule out false positive recombinant clones.
References
1. Boularand, S., Darmon, M. C., and Mallet, J. (1995) The human tryptophan hydroxylase
gene: An unusual complexity in the 5′ untranslated region. J. Biol. Chem. 270, 3748–3756.
2. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high
yield from λ bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220.
3. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991) “Touch
down” PCR to circumvent spurious priming during gene amplification. Nucleic Acids
Res. 19, 4008.
4. Abe, K. (1992) Rapid isolation of desired sequences from lone linker PCR amplified cDNA
mixtures: Application to identification and recovery of expressed sequences in cloned
genomic DNA. Mammalian Genome 2, 252–259.
5. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [SSPE, p. B.13,
SSC, p. B.13, Denhart’s solution, p. B.15, Alkaline gel electrophoresis, p. B.23].
6. Blumberg, D. D. (1987) Creating a ribonuclease free environment. Methods Enzymol.
152, 20–24.