VISUALIZATION AND ANALYSIS OF PCR PRODUCTS IN AGAROSE GEL ELECTROPHORESIS Flashcards
(35 cards)
is a method of separating DNA fragments by movement through a gel-like substance called agarose.
Gel electrophoresis
Derived from a seaweed polysaccharide,________ form small pores that act as sieves to separate DNA based on_____;
whereby____ DNA molecules move through the pores faster and easier than____ molecules.
agarose gels
size
smaller
larger
are oriented at the top of the gel to allow for precise insertion of PCR products into the gel.
Loading wells
An electrical current is applied to move_________ away from a negative electrode (-) and toward a positive electrode (+).
negatively charged DNA molecules
T or F
DNA migrates through the gel in a single, vertical lane.
True
Three factors influence the speed of movement:
(i) the voltage of the electrical field, (ii) the concentration of agarose, and
(iii) most importantly, the size of the DNA molecule.
This laboratory activity will determine the presence or absence of previously amplified DNA in the samples as well as ascertain if the ones amplified by PCR is the actual target DNA or not, through visualization of bands on an______.
agarose gel
________(e.g. bacterial DNA), if present, will be distinguishable based on the size, or base pair (bp) length, of the DNA molecule.
However, DNA itself is not visible within an agarose gel. Thus, a________ is added to the gel that binds DNA and fluoresces under_____.
DNA, which appear as a horizontal line, or band, on the agarose gel, is visualized using either a_____ or ____ (e.g. Gel Doc EZ imager).
Target DNA
fluorescent stain
UV or blue light
UV trans-illuminator or an imaging system
Key Elements for Gel Electrophoresis
- PCR Product
- DNA ladder
- Agarose gel
- DNA stain
- Running buffer
- Loading dye
- Electrophoresis system
The final copies of the target DNA created during a PCR reaction;
the amplified DNA
PCR Products
is a cocktail of synthetic DNA fragments with pre-determined sizes (in bp).
DNA Ladder
The ladder, also called a______ or ______, is loaded alongside experimental samples as a reference tool for estimating band size.
DNA marker or molecular ladder
Agarose Gel
_______is dissolved into ______and boiled until the solution becomes_____.
After slightly cooling, it is poured into a_____ with _____ to solidify and form the agarose gel.
DNA wIl migrate through the gel and form separate bands based on size (correlating to length in bp).
Agarose powder
running buffer
clear
casting tray with combs
is added to the agarose gel to visualize DNA under a______.
DNA Stain
UV or blue light
There are two primary methods of DNA Staining:
a. ________: DNA stain is added to the agarose gel mixture after melting, but before pouring, the gel.
b._________: The gel is incubated in a stain solution after gel electrophoresis.
Pre-stain
Post-stain
is a conductive liquid that allows the DNA to migrate through the agarose gel.
It is important that the agarose gel be made using the same buffer.
Running buffer
Buffers commonly used (3)
Triss/Borate/ETDA (TBE) or
Tris/Acetic Acid/EDTA (TAE) are commonly used.
There are two primary methods of DNA Staining:
a. ________: DNA stain is added to the agarose gel mixture after melting, but before pouring, the gel.
b._________: The gel is incubated in a stain solution after gel electrophoresis.
Pre-stain
Post-stain
Loading dye has two primary components:
i) a________ indicates how far the DNA has run on the gel and
(ii)______, which is denser than the buffer, ensures that samples fall into the loading wells rather than float back out.
Some Taq Master Mixes (e.g., Promega Go Taq) already contain a pre-mixed loading dye.
visible dye
glycerol
Electrophoresis System
_____ and______will be placed into the chamber of an electrophoresis system.
After loading the samples, an electric current is applied to move the________DNA towards ______
Without this electric field, the DNA will not migrate through the agarose gel.
It the electric field is reversed, the DNA will run in the opposite direction, towards the top of the gel, and eventually exit the gel.
Running buffer and the agarose gel
negatively charged DNA towards the positive end of the system.
Pre-Lab Preparation:
_______ Buffer
To prepare 1L of 10x TBE buffer, dissolve 108 g of Tris base and 55 g of boric acid in 900 ml of double distilled water, ensuring the mixture is well combined.
Add 40 mL of 0.5M EDTA, and adjust the final volume to 1L with double distilled water.
Store the buffer at room temperature until use.
10x TBE Buffer
Note:________ is usually concentrated in a 10X or 20X solution.
Dilute the buffer following manufacturer’s specifications using de ionized (DI) or distilled water, not tap water.
Running buffer (commonly TBE or TAE)
Procedure:
1. Remove all unnecessary items from your lab station.
2. Put on______ and clean all surfaces with______.
3. Prepare the sterilized materials needed for Agarose Gel Electrophoresis.
nitrile gloves
70% Ethanol
Prepare Running Buffer Working Solution (0.25% TBE)
- To create a 0.25x TBE solution, dilute__________ in_____ double distilled water for a total volume of 1000 ml..
Note: Use_____. of this solution for gel preparation, and reserve the remaining______ as the running buffer for gel electrophoresis.
25 mL of 10x TBE buffer
975 mL
100 mL
900mL
