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Science & NatureLab 4 A.G.E
By derrickousley
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What: Agarose Gel Electrophoresis (A.G.E.) of PCR products from amplification of yeast genomic DNA with 18SrRNA and Ade2for/Ade2rev primers. (1) assaying DNA purity and quality, and (2) estimating the size of a DNA fragment against known standards. Why: The PURPOSE of this very basic technique of agarose gel electrophoresis is (1) to resolve or fractionate DNA molecules into discrete size classes, and (2) to assess the DNA’s purity.This fractionation or separation of molecules happens for 2 reasons: DNA's net negative charge and net negative charge Materials: Fisher Gel Rig system (buffer tank, gel-casting tray, 10-well comb),

         
Part 1: Setting up the Gel Rig and Pouring the Agarose gel
Remove one Fisher Gel Rig system from the drawer
 
Part 2: Preparing samples and loading gel
retrieve the (a) PCR tube rack, (b) 4 sterile 0.5ml tubes and (c) the microfuge tube rack from lab drawer. Place the 0.5ml tubes in the rack and label the lids exactly as you did for your PCR tubes from last week (eg 1.1, 1.2,1.3,1.4 for group 1).









Assemble the gel-casting tray and comb in the buffer tank
YES
is the gel-casting tray clean and dry?
NO
rinse with RO water and dry with a Kimwipe. Wear gloves at all times.
Follow the table in the Protocall
bring (a) its microfuge rack and (b) PCR tube rack to the instructor’s bench and retrieve a 0.5 ml tube of 1X loading dye (LD) and the Lab#3 PCR reactions from the instructor’s bench. Also retrieve one tube of DNA marker size standards(M) per gel.









retrieve a bottle* of molten agarose from the 60oC water bath and wipe the excess water from the bottle with a paper towel.
Pour ALL of the agarose onto the casting-tray with properly inserted comb
briefly spin (<3 sec) your tubes in the microfuge to deposit all droplets to the bottom; be sure to balance the tubes in the microfuge, and be sure the 0.5ml tubes are placed in the rotor adaptors.
NO
Flick each tube with your fingers, OR vortex very briefly, OR gently pipet up and down with a P-10. Did all droplets go to the bottom?
 







YES


The gel must be allowed to solidify for at least 20 minutes.
Immediately take the empty bottle to the south wall sink and fill it with RO water from the white spigot.
 
Part 3: Load Samples and Run Gel
Remove the casting tray with the gel from the rig and turn it so the gel ends span the buffer compartments.









*Molten agarose gel solution: 35 mls of 1.2% agarose in 1X Lithium Borate buffer(1XLB) with 3.5 ul of GelRed (nucleic acid stain) added and maintained at 60oC in a water bath; how much agarose should be added to 35 mls of buffer to make a 1.2 % agarose solution?
 
 
remove the comb to reveal sample wells under the buffer.
pour running buffer (1XLB) into the compartments until the buffer just covers the gel by a few millimeters abou 300ml









 
 
Note: Load 8ul of the DNA marker in the center lane between the lab group samples.
load the gel with samples from 2 adjacent groups in numerical order with one lane in the center loaded with DNA markers. (follow the table)
deposit the sample into the well without spilling the sample into any other wells and not to disturb the sample layer with a puff of air at the end of loading. Using a P-10 or P-20 pipet set to 8 ul, gently and slowly deposit all 8 ul of each prepared sample into its designated well. Be sure to lift the tip out of the well before you release the plunger on the pipet so you do not suck the sample back into the tip. Also, change pipet tips in between each loading. Load the assigned four wells with your PCR samples, and one in the middle of the gel between the 2 groups’ samples with the DNA size standards(M).









rinse the buffer reservoir and casting tray in RO water and place inverted on paper towels to drain and dry. Record all data and pictures (samples analyzed, estimated sizes determined, type of gel procedure, etc.) into your lab notebook for later incorporation into the final lab report. Compare your estimated fragment sizes with the in-Silico PCR results.
Carefully place the gel-tray with gel directly on the trans-illuminator in the BioRad Gel Documentation system. Approximate your band concentrations against the figure. Analyze your results in terms of concentration and purity; estimate the sizes of your DNA samples.
Remove gel lid with electrodes go to south wall sink with the RO water spigot. remove gel casting tray/gel with and rinse with a gentle stream of RO deionized water
As soon as the dye runs about 2/3 of the gel, turn the power-pack off.
Attach the lid being sure to place the red electrode (the positive or anode) on the side of the gel farthest from the wells. The gels will be run at 110-130 volts, until the fastest moving dye runs into the lowest third of the gel (between 40 minutes and 1 hour; adjourn to Computer Lab for Eukaryotic Gene Exercises).