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131A_B LAB TALK.2

Course: CHEM 132L, Fall 2010
School: San Jose State
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1. PCR Denaturation (94C) Top strand 5'-...............acatcactgg agaaagtctt atgaaactcg cc ..................... gaacga actggtttaa tcttgccgct cccctgcatt..................-3' 3'-...............tgtagtgacc tctttcagaa tactttgagc gg ..................... cttgct tgaccaaatt agaacggcga ggggacgtaa...............-5' Bottom strand 2. Annealing (At least 5C below the TM) Top strand 5'-...............acatcactgg agaaagtctt...

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1. PCR Denaturation (94C) Top strand 5'-...............acatcactgg agaaagtctt atgaaactcg cc ..................... gaacga actggtttaa tcttgccgct cccctgcatt..................-3' 3'-...............tgtagtgacc tctttcagaa tactttgagc gg ..................... cttgct tgaccaaatt agaacggcga ggggacgtaa...............-5' Bottom strand 2. Annealing (At least 5C below the TM) Top strand 5'-...............acatcactgg agaaagtctt atgaaactcg cc ..................... gaacga actggtttaa tcttgccgct cccctgcatt..................-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-...............tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa...............-5' Bottom strand 3. Elongation (72C optimal temperature for Taq polymerase) Top strand 5'- ............... acatcactgg agaaagtctt atgaaactcg cc .................. gaacga actggtttaa tcttgccgct cccctgcatt ...............-3' A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-............... tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa ............ -5' Bottom strand 2nd Round PCR 1. Denaturation 5'end of gene: Top strand 5'- ...............acatcactgg agaaagtctt atgaaactcg cc ..................... gaacga actggtttaa tcttgccgct cccctgcatt..................-3' 3-'A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'- ...............tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa............... -5' Bottom strand 2. Annealing Top strand 5'- ...............acatcactgg agaaagtctt atgaaactcg cc ..................... gaacga actggtttaa tcttgccgct cccctgcatt..................-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-...............tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa............... -5' Bottom strand 3. Elongation Top strand 5'- ............... acatcactgg agaaagtctt atgaaactcg cc .................. gaacga actggtttaa tcttgccgct cccctgcatt ...............-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctg 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-gacc tcttcctagg tactttgagc gg ........................ cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-............... tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa ............ -5' Bottom strand 3rd Round PCR 1. Denature 5'- ............... acatcactgg agaaagtctt atgaaactcg cc .................. gaacga actggtttaa tcttgccgct cccctgcatt ...............-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-A............ tgtagtgacc tctttcagaa gg tactttgagc .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-Agacc tcttcctagg tactttgagc gg ........................ cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-............... tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa ............-5' 2. Anneal 5'- ............... acatcactgg agaaagtctt atgaaactcg cc .................. gaacga actggtttaa tcttgccgct cccctgcatt ...............-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-Agacc tcttcctagg tactttgagc gg ........................ cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC ........................ gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC-3' 3'-............... tgtagtgacc tctttcagaa tactttgagc gg ........................ cttgct tgaccaaatt agaacggcga ggggacgtaa ............ -5' 3. Elongate 5'- ............... acatcactgg agaaagtctt atgaaactcg cc .................. gaacga actggtttaa tcttgccgct cccctgcatt ...............-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-Agacc tcttcctagg tactttgagc gg .................. cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-A............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgtc TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-Agacc tcttcctagg tactttgagc gg .................. cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-Agacc tcttcctagg tactttgagc gg .................. cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-Agacc tcttcctagg tactttgagc gg .................. cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacga actggtttaa tcttgccgct cccctgcatt ............A-3' 3'-............... tgtagtgacc tctttcagaa tactttgagc gg .................. cttgct tgaccaaatt agaacggcga ggggacgtaa ............-5' 5'-CTGG AGAAGGATCC ATGAAACTCG CC .................. gaacag actggtttaa gaattccgct cccctgA-3' 3'-Agacc tcttcctagg tactttgagc gg .................. cttgct TGACCAAATT CTTAAGGCGA GGGGAC-5' What is the expected size of the PCR product? If this plasmid is used, what enzyme(s) should you use for digest? How many cuts will there be? What will be the size of the fragments? If this plasmid is used, what enzyme(s) should you use for digest? How many cuts will there be? What will be the size of the fragments?
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