L3 - Lab 3 - Transformation & Enzymes

L3 - Lab 3 - Transformation & Enzymes -...

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Unformatted text preview: Transformation and enzymes Lab Manual pgs 30-56 (35-42), 11-14, 75-93 Campbell pgs 68-74, 77-86, Ch. 8 Office Hours (2084 VLSB) Mikes: M 11-12, W 10-11, 1-3 (2084/2088) • Lab GSIs: one hour before each lab except W/F 9-12 lab. Also M 2-4. 2084 VLSB • Disc. GSIs: M-F 10-2. 2084 VLSB • Dr. Schlissel: F 9:30-11 in 499 LSA Constructing recombinant DNA 1) Isolate DNA- source (foreign) DNA and vector DNA (source DNA has gene of interest: GOI) 3) Restriction digestions - generate smaller pieces 5) Ligation reaction - insert foreign DNA into vector DNA 7) Transformation - introduce recombinant DNA into host 9) Screen - look for transformed cells with gene of interest 11)Analysis. Note slides 3-19 of this lecture are identical to slides 46-62 of the first lab lecture. Screen: Amp R !-gal Objective: clone a Gene of Interest (G.O.I.) Anneal & Ligate Isolate & Digest Transform & screen Weak Strong DNA structure - essential to understand! Transform & Screen Ligate: 3 possible results No insert! Insert but not GOI. Insert is GOI. Restriction digestions Mix & Anneal AATT TTAA AATT TTAA Multiple pieces! = sticky end Transform & Screen Restriction digestions Mix & Anneal AATT TTAA AATT TTAA Multiple pieces! = sticky end Your experiments- only one type of DNA. This means any insert is your GOI. 10,000 20,000 30,000 40,000 48,502 LAMBDA Bam H1 SITES 5505 22,346 27,972 34,499 41,732 5505 16,841 5626 6527 7233 6770 21,226 21,226 26,104 31,747 39,168 44,972 4878 5643 7421 5804 3530 Eco R1 SITES Hind III SITES 23,130 25,157 27,479 36,895 37,459 37,584 44,141 23,130 2027 2322 9416 564 125 6557 4361 Restriction Maps of the Linear Genome ! ! ? •5’------------- AAGCTT-- AAGCTT-------- 3’ •3’------------- TTCGAA-- TTCGAA-------- 5’ Anneal & Ligate Isolate & Digest Transform & screen Already done for you. Student exercise. Restriction sequence for Hin D III • 5’ AAGCTT ---- AAGCTT ----- AAGCTT 3’ • 3’ TTCGAA ---- TTCGAA ----- TTCGAA 5’ Piece 1 Piece 2 • 5’ AAGCTT ---- AAGCTT ----- AAGCTT 3’ • 3’ TTCGAA ---- TTCGAA ----- TTCGAA 5’ Piece 3 Piece 4 Sticky ends & annealing • 5’CA AGCT T ---- A AGCT T ----- A AGCT TCCG 3’ • 3’GT TCGA A ---- T TCGA A ----- T TCGA AGGC 5’ Piece 1 Piece 2 Piece 3 Piece 4 Possible results? Ligation- sealing the ends!...
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This note was uploaded on 09/28/2009 for the course BIO 1al taught by Professor Pederson during the Fall '08 term at Berkeley.

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L3 - Lab 3 - Transformation & Enzymes -...

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