Agarose Gel Visualization of Restriction Enzyme Digest
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Transcript of Agarose Gel Visualization of Restriction Enzyme Digest
BRIDGES 2014
Agarose Gel Visualization of Restriction Enzyme Digest
Gel Electrophoresis: Main Principles
Separate DNA fragments by size Smaller DNA fragments move faster Run a “size marker” to compare size of separated
fragmentsDNA moves through gel due to electric field
DNA is negatively charged Moves towards positive charges
Visualize separation Use nucleic acid stains such as Gel Red or ethidium
bromide When excited under UV light transmits at visible
wavelength
Electric field causes DNA to move
Smaller Fragments Move Farther
100bp size marker
Size markers tell how big our fragments are
Agarose concentration affects speed of movement
Visualize using stains and UV light
Where the magic happens
Step 1: Pouring a gel
Need to create gel of appropriate agarose concentration Combine agarose (powder) TBE (liquid buffer)
Microwave to dissolve agaroseAdd stain and swirl to mix
Gel RedPour into gel bed
With a comb!
Preparing a 2% agarose gel with 50mL 1x TBE
Weigh out 1g agarose in weight boatAdd 50ml of 1x TBE
Needs to be diluted!Cover with saran wrap
Poke holes!Microwave til it boils
Careful it’s hot!Allow to cool to room temperatureAdd 5ul Gel RedSwirl to mixPour into gel bed Insert comb Let solidify
Loading a gel
This is easy but takes practice So practice!
The gel will be submerged in bufferTip does not need to go INTO wellHold tip over well and release!
Loading dye is heavy it will fall into placeTry not to poke holesMake a list of what sample goes where BEFORE
loading gelMake sure we are loading at the NEGATIVE end
Running a gel
DNA needs electric field to movePower source supplies this electric fieldSet power source to the voltage you want and
let run for specified amount of timeMake sure positive goes to positive and
negative to negativeMake sure wells are at negative end!
Loading and Running a Gel
Take out comb Make sure wells are at negative end Fill gel rig with 1x TBE buffer
Enough so wells are well covered Make a list of what sample will go where Load:
20 µL of pBR322/BstNI size markers 10 µL of the undigested (U) sample/loading dye mixture 16 µL of the digested (D) sample/loading dye mixture
Carefully release this while holding tip OVER the well Sample will fall into well
When finished put lid on gel bed (+/+ and -/-) Hook up to power source Set power source to 200v and start Check in- did the bands move?
Visualizing Gels
Gel Red attaches to DNA Intercalates between base pairs
“Slips in” This strips away water
Gel Red glows under UV light Removal of water during intercalation allows it to
glow brighter
Analyzing Results
Looking at banding patterns Bands present? Number of bands per each lane? Relative size of bands
Using 100bp ladder
Visualizing Gels
Turn off power sourcePour running buffer back into containerTake gel out of gel rig
Be careful not to break it!Bring to UV transluminatorVisualize
Make sure you are wearing a shield!Take photos and analyze