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Questions lab 2

Questions lab 2 - μ mol/10 6 pmol x 10 6 μ l/L = 20 μ M...

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Questions lab 2: 1. Please locate each primer within the gene sequence and determine how many nucleotides were changed. Draw the primer sequence and the strand to which it will anneal. Where is the start codon relative to the 5’ primer? Where is the stop codon relative to the 3’ primer? 2. What is the final concentration of dNTPs and of MgCl 2 in your PCR reactions? Show the calculations. 100 mM x 1 μ l=c x 50 μ l c= 2 mM 25 mM x 5 μ l=c x 50 μ l c=2.5 mM 3. How many units of Pfu polymerase were present in your PCR reaction? 10 u/ μ l x 0.5 μ l = 5 units added in the total volume. 4. What is the concentration of the 5’ primer stock (20 pmol/ μ l) in μ M? Show the math. 20 pmol/ μ l x 1
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Unformatted text preview: μ mol/10 6 pmol x 10 6 μ l/L = 20 μ M 5. What is the recognition site for each restriction enzyme you used (look this up at www.neb.com)? When the restriction enzyme cuts the DNA, what will the ends look like? Does it leave a blunt end, a 5’ overhang, or a 3’ overhang? Draw this out. NdeI leaves 5’ overhangs as do all the enzymes we used: 5’CATATG3’ 5’CA3’ 3’GTATAC5’ 3’ GTAT5’ 6. How would you make 150 ml of a 1.2% agarose gel? 1.2% = 1.2 g / 100 ml 1.2g x 150/100 = 1.8 g 1.8 g of agarose in 150 ml of 1x TAE, boil, cool, pour 7. What sized PCR product do you see? What is the size of the digested plasmid vector?...
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