By Michael A. Innis, David H. Gelfand, John J. Sninsky, Thomas J. White
The wide software of the polymerase chain response (PCR) technique is now in the succeed in of each researcher. Designed to be used on the laboratory bench, this is often the main finished handbook on PCR on hand this present day. Over 50 chapters offer specific directions on strategies, with suggestion on primer layout. the entire options defined, from amplification and direct sequencing of genomic DNA via cDNA cloning and quantitation of mRNA, are proven, present, and supplemented with worthwhile notes and illustrations. you are going to additionally learn the way to:* Optimize novel purposes* stay away from many cumbersom. Read more...
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Extra info for PCR Protocols. A Guide to Methods and Applications
On the other hand, blunt cloning requires fully extending and kinasing the P C R products and is inefficient. A further increase in the frequency of the desired product can be obtained by directional cloning of the RACE products after cleavage with appropriate restriction enzymes. By cleaving the RACE prod ucts with two endonucleases, each specific to a different primer, the cloning of any nonspecific amplification product that has the same primer at both ends will be precluded. Directional cloning can be used to obtain an even higher frequency of the desired product, providing that a restriction enzyme that recognizes infrequent sites can be used to cleave the specific product at a site internal to the gene-specific primer.
B. McCabe. 1988. Synthesis of hexokinase I (HKI) c D N A probes by mixed oligonu cleotide primed amplification of c D N A (MOPAC] using primer mixtures of high complexity. Amer. f. Hum. Genet. 4 3 : A 1 8 5 and in Biochem. Med. Met. Biol. Manuscript in press. Lathe, R. 1985. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. /. Mol. Biol. 1 8 3 : 1 - 1 2 . Lee, C. C , X . Wu, R. A. Gibbs, R. G. Cook, D. M. Muzny, and C. T. Caskey. 1988.
The 32 guessmer o l i g o n u c l e o t i d e p r o b e was e n d labeled [ P ] A T P (3000 C i / m m o l ) guessmer in t h e p r e s e n c e of b y T4 p o l y n u c l e o t i d e k i n a s e . T h e p r o b e h y b r i d i z a t i o n w a s p e r f o r m e d at 4 2 ° C i n 6 x SSC, 0 . 1 % s o d i u m p y r o p h o s p h a t e , 0 . 1 % SDS, 0 . 5), a n d 50 /xg/ml o f d e n a t u r e d h e r r i n g s p e r m DNA. T h e b l o t w a s w a s h e d i n 2 x S S C , 0 . 1 % SDS at 4 2 ° C . 50 Part One.
PCR Protocols. A Guide to Methods and Applications by Michael A. Innis, David H. Gelfand, John J. Sninsky, Thomas J. White