By Ralph Rapley
A good, complete selection of the main trustworthy protocols for PCR sequencing-all constructed and defined through eminent researchers and scientists who use them effectively each day. those protocols deal virtually with these difficulties accompanying the swift reassociation of amplified complementary strands through displaying how sequencing reactions should be successfully changed, in addition to complicated equipment of primer manipulation that permit the affinity purification, or its transcript construction, of homogeneous unmarried strands. additionally they supply the entire worthy info of PCR purification, primer labeling, the training of sequencing gels, in addition to of traditional equipment for cloning PCR items into vectors and their next sequencing.
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Extra info for PCR Sequencing Protocols
5 pL) c. [cx-~~~] dATP d. Sequenase 3 U from United States Biochemicals 2. Total volume should be approx 15 pL; mix thoroughly and incubate for 2-5 min at room temperature. 3. Termination Reactions 1. 5 PL of the appropriate dideoxy termination mixture. 2. 5 pL of the labeling reaction to each of the other three tubes (“A,” “T,” and “C”). 3. After 2-5 min of incubation at 37’C, add 4 PL of stop solution to each termination reaction, mix, and store on ice. 4. To load the gel, heat the samples to 75-gO°C for 2 min, and load 2-3 PL in each lane.
The following procedure has been used to sequence the control region of human mitochondrial DNA and utilizes a gel purification step because of the relatively inefficient amplification of the DNA with 20 @ dNTPs in the PCR. Spurious PCR products can also interfere with the reading of the DNA sequence; gel purification removes these products from the fragment of interest. Other PCR products can be effictently sequenced without the gel purifica- tion step (I) but this step is included for completeness should it be required.
And Maryanski, J. L. (1992) H-2 restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity. J. Exp. Med. 176,439-447. 4. , and Maryanski, J. L. (1993) T cell receptor selection by and recognition of two class I MHC restricted antigenic peptides that differ at a single position. J. Exp. Med. 177,81 l-820. 5. , Maryanski, J. , and Luescher, I. (1993) Differential TCR photoaffinity labeling among H-2Kd restricted CTL clones specific for a photoreactive peptide derivative.