Biotech W16-Lab day 4-week 2-protocol quiz Flashcards
For the determination of biotin labeling efficiency, what are samples A, B and C?
A - commercially prepared biotin-labeled DNA
B - control reaction made for us using the same buffers and enzymes
C - the student prepared biotin-labeled probe
What sample is kept (between A, B and C)?
C
What are the dilutions that are made for each of the samples?
1: 10
1: 100
1: 500
What is the commercially prepared biotin labeled DNA and how is it diluted?
Fermentas DNA. Add 1 uL of it and 9 uL of water for the first dilution.
What is the test blot membrane made of?
nylon
What is important when handling the membrane?
wear gloves and only touch the edges.
What volume of each of the diluted samples is added to the membrane?
1 uL
How many grids should there be, and how are these drawn, on the membrane?
12 and with a permanent marker
How long is the test blot allowed to dry?
15 minutes and air dried
What is done to ensure that the DNA samples are not washed off the membrane?
Cross-linking of the DNA samples to the GeneScreen Plus membrane
What is the desired program in the GS gene linker?
(C-L: 125 ms)
The membrane is placed on a paper towel and placed where?
in the Bio-Rad GS gene linker
AT the end of the program, what will be heard (i.e. after reaching the set time of the set energy level)?
A tone for a few seconds
How can the tone be stopped?
By pressing any button
To begin the chemiluminescence reaction (i.e. to get it prepped, not run it), what is done first?
Add 10 mL of blocking solution too a hybridization bottle.
What is the blocking solution?
1X TBST + 1% skim milk
What is the purpose of the blocking solution?
The blocking solution utilizes a inert protein to prevent non-specific interaction of the antibodies.
How is the test strip transferred to the tube?
Using clean forceps.
How long is the strip with the washing solution added to the rotisserie (fkn top kek)?
15 minutes at room temperature
How is the membrane washed?
Decant the blocking solution and wash the membrane with 10 ml of 1X TBST. Allow to wash for 5 minutes.
During the first wash, what is done?
Prepare stabilized streptavidin peroxidase conjugate.
How is the stabilized streptavidin peroxidase conjugate prepared?
Add 10 uL of streptavidin peroxidase conjugate to a 10 mL fresh aliquot of 1X TBST + 1% skim milk and vortex for 2 seconds to mix.
After decanting the wash buffer, what is added to the test strip?
The streptavidin horseradish peroxidase conjugate.
How long is the test strip and enzyme conjugate incubated for on the rotisserie?
25 minutes at room temperature
What is the purpose of using streptavidin?
It has a very strong affinity for biotin (as you may have guessed)
After the 25 minute incubation, what is done?
Pour of the conjugate solution and wash with 10 mL of TBST for 5 minutes. (bis = latin repeat)
AFter the second washing step, what is done?
Remove the test strip from the buffer and place into a large weigh dish. add TBST
What is the purpose of adding TBST after the wash step is complete?
Keep the membrane from drying out.
How is the “light” image taken?
Place the test strip in the imager using forceps and acquire the image.
What is done for the chemiluminescent reaction?
Do not move strip after taking “light” image.
Mix luminol and oxidizing agent, invert gently to mix but not make bubbles.
Pour the mix on top of the test strip and incubate for 1 minute.
Set the syngene imaging system to chemiluminescence and capture an image of the test strip with the overhead light off.
