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