By Peng Liang
Drs. Peng Liang and Arthur B. Pardee gather for the 1st time a finished assessment of the cutting-edge in their strong new method and functional purposes. The book's pioneering individuals describe all of the significant parts of this novel know-how, together with either RAP-PCR and DD utilizing fluorescence detection, in addition to their strong approach for choosing and cloning family-specific genes. additionally they offer various examples within which differentially expressed genes have been effectively pointed out in various organic platforms starting from crops to songbirds to people.
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Extra info for Differential Display Methods and Protocols
1 PREPARATION OF THE HAP COLUMN 1. Weigh out about 1 g of hydroxyapatrte (HAP) mto a 50-mL conical tube. This should be about 7 5 mL of dry HAP Add 50 mM phosphate buffer for a total volume of 45 mL and swirl gently Let HAP settle for 10 mm, then pipet off the cloudy upper layer containing the “fines,” and repeat this procedure until the buffer above the settled HAP does not appear as cloudy 2 After asprratmg off the buffer for the last time, then add an equal volume of 50 mM phosphate buffer to the HAP slurry.
2. Identification of an FGF- 1-inducible mRNA by differential display. RNA isolated from quiescent or FGF- 1-stimulated NIB-3T3 fibroblasts was converted to cDNA using random hexamer primers. PCR was then performed using a degenerate sense zinc finger (ZF) primer and a degenerate antisense protein tyrosine kinase (PTK) primer (top panel). As a control for the cDNA synthesis, PCR was also performed using sense and antisense FGF receptor (FGFR)- 1 primers (bottom panel). Amplification products were displayed using agarose gel electrophoresis and ethidium bromide staining.
The asterisks mark some of the bands representing mRNAs with putative increase or decrease in expression after treatment. 5-mL thin wall PCR tube. Perform PCR using the following program: (94°C for 20 s, 42°C for 20 s, 72°C for 30 s) for 40 cycles at 72°C for 10 min (we use the GeneAmp PCR system 9600 machine from Perkin Elmer). 2. L sequencing loading dye, heat to 80°C for Smin and load on a 6% acrylamidelXA4 urea sequencing gel (load the duplicate reactions for each RNA condition in adjacent lanes) (see Note 2).
Differential Display Methods and Protocols by Peng Liang