Question

Design a PCR experiment to amplify a sequence of interest. Use the following reagent concentrations for calculating your cocktail. Fill in the chart below.

A. Fill in the chart below with the volumes that would be added to each tube. Note the final volume of the reaction. Show your calculations for full credit.

REAGENT	Final Concentration (50 µl Reaction)	Test Reaction (+)	Control Reaction (-)
10x Reaction Buffer	1X	mL	mL
dNTPs (15 mM)	200 µM	mL	mL
Forward Primer (5 mM)	0.2 µM	mL	mL
Reverse Primer (5 mM)	0.2 µM	mL	mL
Taq DNA Polymerase 4 units/µl	2 units	mL	mL
Template DNA (150 ng/µl)	100 ng	mL	mL
Sterile, distilled water	calculate	mL	mL
TOTAL VOLUME		50 mL	50 mL

Answer 1

Answer Buffer= V V=RT where, R=Reqd concentration G=glven conc. G (504) So, we have to find out v far each care. Far #1= v= 1:0 = 10x50 = 5ml simillarly we have to find rest of the v.

Final concentration Do ul Reaction . Test Reacion to me I control Reaction Elme 5ml . 5 ml lx Reagent 10x Reaction buffer dNTPs (15mm) Farward Primers (5mm) 2004M 0.66ml 0.Gome 0.24M 2ml zmy Reverie Primers (5mm) 0.24M ame 2ml 2uk - 1 ml 1 ml Add water). Tag DNA polymerase (4 mit/41) Template DNA (150ng/ul). Sterile distilled water 100 ng. 2- 3ml 36-4 ml I (NODNA) 50ml TOTAL VOLUME