Lab 5 - Lab 5 Cloning Plasmid isolation Restriction...

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Lab 5 Cloning Plasmid isolation Restriction digestion Sequencing Complementation Vibrio isolation Mitosis vs. Meiosis Answer keys for pre-labs and worksheets are posted in the hallway! Come to office hours in VLSB 2084!
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RNase NaOH+SDS Chromosomal DNA plasmids LB culture Analysis : Plasmid isolation What is in the buffers? What is the function? Analysis
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Analysis : Restriction Digestion Amplification: 1) E. Coli divide 2) ORI Hind III Hind III Hind III
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Restriction enzymes recognize specific sequences (often palindromes) and cut two phosphodiester bonds in double stranded DNA. Restriction enzymes have host defense function in bacteria. They protect endogenous DNA from being cleaved by adding methyl groups to their DNA (methylase). 25% A = 25% T so 25% G and 25% C Probability (p) that there is one 1 A in the sequence is ¼. p for ATCGAT = ¼x±¼x±¼x±¼x±¼x±¼ = (¼) 6 Fragment size generated = 1/p = 1/ (¼) 6 = 4 6 Genome has 3 billion bp. How many fragments Generated when cut with a six base pair cutter? 20% A = 20% T so 30% G and 30% C Probability (p) that there is one A in the sequence is 1 / 5 . p for one G would be 1.5 / 5 p for ACTAGT = 2 / 10 x 3 / 10 x 2 / 10 x 2 / 10 x 3 / 10 x 2 / 10 Fragment size generated = 1/p 1/( 2 / 10 x 3 / 10 x 2 / 10 x 2 / 10 x 3 / 10 x 2 / 10) Do the math for this 6 base pair cutter.
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Analysis : Gel electrophoresis (separation by size + charge ) Next Week! Phosphate groups = negative charges Migrates to Anode athode Anode Increase resolution by increasing amount of gel matrix (e.g. 2% vs 1% agarose) Analysis of big fragments requires low % gel Analysis of small fragments requires high % agarose or acrylamide
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Analysis : DNA Sequencing Important: Direction Of Synthesis
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4 tubes: All contain dNTP, primer, DNA polymerase, buffer, Each just 1 type of ddNTP Primer initiates DNA synthesis by DNA polymerase Ratio of dNTP : ddNTP is important!!! Normal 300 dNTP : 1 ddNTP dNTPs contain 3’ OH (can be elongated) ddNTPs contain just H (terminate!) Sequence of the synthesized strand G A C T G A A G C T G T T 3’ 3’ 5’’ Template molecules
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Start with 1,000,000 molecules template DNA and DNA polymerase, primer and buffer dCTP:ddCTP is 90:10 dATP:ddATP is 1:101 dGTP:ddGTP is 50:50 dTTP and ddTTP were completely forgotten You need 10,000 molecules to show up in the gel Will you see bands? How many? Make up a sequence and draw a gel. .. Do the math! Examples in the Exam reader! ddCTP tube : 90 dCTP (1,000,000/100 = x/90) : 10 ddCTP (1,000,000/100 = x/10) 1) 900,000 molecules incorporate a dCTP and can be elongated 100,000 molecules incorporate a ddCTP (no 3’OH = terminate), since > than treshold = band 2) 900,000/100 = x/90; 900,000/100 = x/10 810,000 molecules incorporate a dCTP and can be elongated 90,000 molecules incorporate a ddCTP (no 3’OH = terminate), since > than threshold = band
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This note was uploaded on 10/29/2009 for the course BIO biology taught by Professor Meighan during the Fall '09 term at University of California, Berkeley.

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Lab 5 - Lab 5 Cloning Plasmid isolation Restriction...

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