Lab 5: Magnesium Titrations Flashcards
What concentrations of MgCl2 are to be set up for PCR reactions?
0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 5.0 mM
What components can be premixed for the PCR reaction?
All components other than H2O and MgCl2
What is the total volume of the PCR reaction?
25 µl
What are the temperature and time settings for the initial denaturation step in PCR?
95 °C for 5 minutes
What are the temperature and time settings for the denaturation step in PCR?
95 °C for 30 seconds
What are the temperature and time settings for the annealing step in PCR?
59 °C for 30 seconds
What are the temperature and time settings for the extension step in PCR?
72 °C for 30 seconds
How many cycles are performed in the PCR protocol?
35 cycles
What is the purpose of adding ethidium bromide to the gel?
To allow visualization of DNA
What is the final concentration of ethidium bromide to be added to the gel?
0.5 µg/ml
What is the procedure to prepare the gel template for electrophoresis?
Seal ends with tape, insert comb, pour agarose mixture
What is the voltage and duration for electrophoresing the agarose gel?
100 V for approximately 30 minutes
How is the DNA sample prepared before loading into the gel?
Add 10 µl of DNA and 2 µl of 6X loading buffer to a total volume of 12 µl
What is the stock concentration of dNTP used in the PCR protocol?
10 mM
What is the concentration of the primer mix used in the PCR protocol?
100 ng/µl
