Question

1.

a) You have a stock tube of 50 mM MgCl₂ and you need to optimize MgCl₂ concentration for the Q8 SYBR Green primers. Create a table to describe how you would create a dilution series so that MgCl₂ could be tested at 5 mM, 4.5 mM, 3 mM, 2.5 mM, 1 mM, 0.5 mM and 0 mM to optimize a 25 l PCR (see the table below). Your new stocks should be in a 100 μL volume.

b) Once you’ve developed your dilution series, complete the PCR master mix table below. When deciding on the number of reactions for your master mix, be sure to account for appropriate controls. Assume your cDNA is 50 ng/uL and your template is the size of Marburg virus genome

Stock	Working		µL of stock for 1 reaction		Master Mix µL of stock for 9reactions
10X PCR buffer	1X
50 mM MgCl2	0-5 mM MgCl2
10mM dNTP	0.8 mM dNTPs
Viral cDNA, 50 ng/μL	107 copies
Forward Primer (5 µM)	0.5 µM
Reverse Primer (5 µM)	0.5 µM
Taq Polymerase (5U/µL)	2.5U/25 µl rxn
H20
Total			25 µL

c) Draw a theoretical agarose gel that demonstrates what you would expect to see from your MgCl2 optimization experiment when you use your Q8 SYBR Green primers. Your gel should have enough lanes to accommodate seven MgCl2 concentrations plus one molecular weight ladder plus any appropriate controls.

Answer 1