Differential Display Methods and Protocols - download pdf or read online

By Peng Liang

ISBN-10: 0896034054

ISBN-13: 9780896034051

1st, 1997, Humana Press. Vanderbilt melanoma heart, Nashville, TN. finished evaluate of this new expertise, together with method and functional purposes. For investigators learning gene expression legislation. Plastic-comb, spiral binding.

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Search restrtctron enzyme sites m the nucleottde sequences of the candidate clones. Digest the amplified mserts of the clones and the origmal FDD reaction with these restrtction enzymes, and run them m parallel to compare the digestion pattern of the target band and the mserts (“restrtctton test”) Select clones that are digested stmilarly to the band m FDD reaction for further analysrs 6 Confirm the expression pattern of the correspondmg transcrtpt by Northern blot hybndrzation, RNase protectton or quantitative RT-PCR assays 4.

5’ primer (lo-mer) (25 CrM) (see Note 1). 3. 01% w/v autoclaved gelatin (Perkm Elmer, Foster City, CA, cat no N808-0006) 4. 20 pA4 dNTP mix 20 pA4 each of dATP, dGTP, dCTP, dTTP 5 200 @4 dNTP mix 200 cuz/ieach of dATP, dGTP, dCTP, dTTP 6 [cz-~~S] dATP (1000 Cl/mmol) (Amersham, Arlington Heights, IL). 7 Ampli Taq 5 U/pL (Perkm Elmer; cat no N801-0060) 8. Sequencing loading dye: 95% Formamide, 20 mM EDTA, 0 05% bromophenol blue, 0 05% xylene cyan01 FF 9. X-ray film: Kodak XAR 5. 10 TA cloning kit (Invitrogen, Carlsbad, CA, cat.

Differential display (DD) (I,21 and subtractive hybridization (SH) (review see refs. 3-5) are by far the most common methods currently used by investigators for this purpose. However, do these techniques identify both abundant and rare mRNAs? Previous kinetic studies (6,7) indrcate that approx 98% of all mRNA species within the cell have a prevalence ranging from l/10,000 to l/100,000, and thus are considered rare. Furthermore, the remammg approx 2% of mRNA species (about 200-500) contribute to over 50% of the total mRNA mass (6,7).

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Differential Display Methods and Protocols by Peng Liang

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