LAB report 12 HOURS( 2 PAGES)

Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis

Objective

· Digest DNA of pGLO plasmid using restriction endonuclease enzymes.

· Run an agarose gel to separate the DNA fragments.

Background

Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid

Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light.

Materials

Restriction digest

· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE)

· Plasmid prepared in lab 7

· NanoDrop Lite spectrophotometer

· Microfuge tubes – Sterile

· 37 C degree bath – block heater

· Sterile 10ul and 200ul tips

· Bleach bottles for cleaning bench

· 10X NE Cut Smart Buffer – comes with enzyme

· Nitrile gloves

· Sterile DI water

· Shaved ice

· Ice block for enzymes

Gel Electrophoresis

· Agarose

· Sterile miliQ Water

· 15 well comb

· 50x TAE buffer

· DNA ladder – diluted in sample buffer (1 KB)

· Gel loading dye

· Gel electrophoresis chamber

· Power supply

· Ethidium bromide

· Gel Sys – visualization system

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Procedure

Restriction Digest of plasmid DNA

· Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide

· Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests.

· Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix.

· Label an Eppendorf tube ‘+’ and another ‘-‘

· Make up a reaction mix in both tubes as follows for one of your plasmid samples

· add 1ug of DNA from your plasmid prep

· 5ul of 10X NE Cut Smart Buffer

· Sterile DI water to make the reaction mix to 50ul

For the + tube

· Add the restriction enzymes last to the + tube ONLY

· Repeat with the other two plasmid samples

For the – tube

· Do not add any enzyme to the ‘-‘ tube

· Repeat with the other two plasmid samples

· Mix the tubes by flicking – DO NOT VORTEX

· Give a 5 second spin in the centrifuge to bring the contents to the bottom

· Incubate for 65 mins in heating block.

· Place the tubes on ice to stop the reaction

Gel Electrophoresis

For one gel

· Block the ends of the gel mold using time tape. Insert the sample comb for 8 or 15 wells.

· Place the tray in the in the plastic lid box to catch any leaks.

· Weigh out the amount of agarose needed to prepare 50 ml of 1% agarose

· Transfer the agarose to a 250ml Erlenmeyer flask.

· Dilute 1 ml of 50X TAE buffer to 50 ml in a graduated cylinder and add to the agarose.

· Place a piece of paper towel in the neck of the flask so it partially blocks the neck.

· Microwave on high for 30 seconds for up to 4 times. USE A SILICONE HANDGRABBER. Swirl flask gently in between microwaving. STOP when the agarose is clear and no more agar powder is visible.

· Cool on bench until it is comfortable to handle.

· Add 0.5ul of ethidium bromide (CAUTION – MUTAGEN) to the agarose and gently swirl.

· Allow gel to cool so it can be placed against the inside of the wrist. Running it under cold water helps, but do not let it set.

· Gently pour the agarose into the template mold and let it set for 10 minutes or more

· Gently remove the comb and the bumpers from the gel.

· Mix 250ml of 1xTAE using your 50X stock

· Place the gel still in the holder in the gel box.

· Pour buffer into the box until it covers the gel.

· Load 10ul of diluted ladder very slowly to the center lane, and the end lanes

· In clean microfuge tubes make up the following.

· For your + and - samples run these in duplicate at different levels

· Load the gel as follows:

· Very slowly add each of the + and – digests either side of the ladder

· Orient the gel so the sample is at the cathode end (black) and the samples will flow in the direction of the arrow on the side of the box.

· Place the top on the box and connect the electrodes and turn the power on.

· Set the voltage to 150 volts and hit run. The display will show 150 volts and you should see gas coming from the electrodes.

· Run until the indicator dye is close to the bottom.

· Move the gel to the Gel illuminator and view the bands.

Questions

· See the assignment in Canvas