Flashcards in Biotech lab week 2-quiz on protocol-Day 3 Deck (21)
What is added initially to the restriction enzyme reaction before the addition of the enzyme?
pROK2 DNA, NeBuffer, ddH20
Before the addition of the enzyme, but after the addition of the precursor reagents, what is done to collect all the liquid at the bottom?
Pipet together DNA, buffer and water and gently vortex on medium speed followed by a brief pulse spin.
What should the total reaction volume be?
When adding the enzyme, it is important to minimize its exposure to?
Temperatures above -20C to prevent deactivation.
The tubes for the restriction digest are incubate for how long and at what temperature?
37C and 1 hour
What is done during the incubation time?
Preparation of a biotinylated probe
What is the first step for the preparation of the biotinylated probe?
Thaw out all the non-enzymatic labeling components.
What are the non-enzymatic labeling components?
cDNA template, 5X reaction buffer, biotin labeling mix, ddH2O
After thawing the non-enzymatic labeling components, where are they placed?
What is done to recover the full volume of the tubes?
How much DNA is added to the 1.5 mL microcentrifuge tube?
How much ddH20 (nuclease free water) is added to the DNA, to reach a total volume of 34 uL, if the DNA concentration is 50 ng/uL?
If the concentration is 50 ng/uL (similar to ours). (for the DNA), then since we added 1 uL of DNA, then:
1 uL/50 ng * 1000ng (required amount of template) = 20 uL.
34 -20 = 14 uL
What is the required amount of template DNA?
What is the volume for the reaction with DNA for the making of the probe?
After adding the water to the DNA, what is added?
10 uL of 5X nucleotide reaction buffer.
Once the nucleotide reaction buffer is added, what is done to the tube?
Vortex and pulse centrifuge to collect all the liquid at the bottom of the tube.
What conditions are used to denature the template?
95C on a dry block for 7 minutes, then place on ice for 3 minutes (Briefly centrifuge)
After denaturing the template, what is added?
5 uL of biotin labeling mix and 1 uL of Klenow reagent (to be added by the GA)
How long is the template biotinylated for?
37C incubator overnight
After the 1 hour incubation, for the restriction digest, what is done?
add 5 uL of 6x DNA loading dye and a brief spin. The tubes are then stored.