Making a Radioactive Probe

Radioactive Hybridization Solution
Setting Up a Blot
Chemiluminescent Blots
Random
100
What is the first Step of making a Radioactive Probe?
Add DNA template probe, water, and nanmers and boil.
100
Name 2 Blocking agents used.
Denhardt's, Yeast RNA, Human Placenta DNA, Herring Sperm
100
After the gel has been imaged, what step can be omitted before denaturation?
Depurination step.
100
Why is there a digested and undigested product?
To determine methylation status.
100
During the 6 wash steps on Day 4 of the Radioactive blot, what happens to the salt and temperature?
Salt decreases and temperature increases.
200
What is the purpose of the 2 hour incubation at 37C (step 3)
To make the complimentary strand of template probe (incorporates the radioactive P-dCTP)
200
Name the two salts in SSC buffer.
Sodium Chloride, Sodium Citrate
200
What is the Denaturation solution?
NaOH
200
What two enzymes are used?
BsaHI and Hpy9991
200
Why do you have to dry (bake) the membrane?
To attach DNA to the membrane, and prevent it from washing off.
300
Why do we add STE at step 4 of making the probe?
EDTA in the solution stops the reaction.
300
Name 2 purposes of Formamide.
Keeps DNA single stranded and lowers the annealing temperature
300
What must be done after denaturation?
Neutralization
300
Why is a special buffer used during PCR?
The product has a high GC content.
300
Why is BSA added to reaction tubes?
Competes with enzymes for binding sites on the tubes (allows the enzyme to go into solution).
400
What is the purpose of the column process?
Removes salt and unincorporated P-dCTPs
400
What is the purpose of Sodium Phosphate?
Keeps pH at 8.0
400
What type of Membrane is used for the DNA transfer?
Nylon membrane
400
What component of the probe allows for signal to be detected later in the process?
Alkaline phosphatase.
400
Why is it important to remove air bubbles when setting up the blot?
Air bubbles will prevent DNA from transferring to the membrane.
500
What is the purpose of the ice bath in the last step?
Quickly lowers the temperature to below the annealing temp (stops reaction energy and keeps single stranded).
500
What does Dextran Sulfate do that concentrates the probe?
Binds the water in the reaction.
500
Why is the gel inverted (upside down) during a transfer?
The DNA is closest to the back of the gel.
500
What binds to the AlkPhos to produce chemiliminescent signal?
CDP-Star substrate
500
What is the purpose of SDS in the hybridization Solution?
Prevents non-specific binding.
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