Lab 2: Learning to work like a Molecular Biologist II: Restriction Enzymes and Agarose Gel Electrophoresis Ruhi Kiflen

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Procedure 1) Label 1.5 mL centrifuge tubes Unknown 1 and Unknown 2 restriction digests will be performed). Label the third tube Master Mix. 2) Make the Master Mix using a fresh yellow tip for each reagent. Final volume should be 15 l. ( Note: 10 units of
Labeling tubes ensures clarity and will prevent from making unnecessary errors.

The Master Mix is created to reduce the tubeto-tube variation that results from pipettor error, when we are using small volumes

enzyme in 1 l)
3) Add 5 l of Master Mix to each
Touching the pipette tip to as near to the bottom of the centrifuge tube will create a capillary reaction to pull solution out of the end of the pipette tip.

unknown tube. When adding, touch pipette tip to the side of the 1.5 centrifuge tube.
4) Add 5 l of DNA from Unknown 1 to

another labeled tube. Pipette unknown DNA into the Master Mix (already at the bottom of the tube). Add another 5 l of DNA from Unknown 2 to labeled tube.
5)

Place reaction tubes in 37 degrees Celsius water bath and incubate for 30 minutes.
6)

The restriction enzymes cleave best at this temperature.

Take your unknowns from the water

bath
7)

Add 1 l of loading dye and 1 l of 100X SYBR Safe DNA stain to each tube containing the digested unknown plasmid
8)

The loading dye will help to weigh down the DNA solution, allowing it to sink to the bottom of wells and not float. It also helps determine relative position of the DNA. The stain helps to see the DNA since DNA is white.

Close the tube top and tap the bottom of the tube gently to mix.

9)

Load 1 lane in the designated gel

with 1 digested unknown plasmid DNA sample 10) Use a micropipettor to load entire contents of your tube containing the digested plasmid DNA (12 l) into a separate well in the gel. a. Steady pipettor over well using two hands b. Depress plunger to push sample to end of tip if there is air c. Dip pipette tip through surface of buffer, centre it over the well and gently depress pipettor to expel sample 11)- 15) TA will perform these steps. PART 2 PROTOCOL 1) 1.0 % agarose gel has been made for you 2) Obtain genomic sample from Lab 1 3) Add 10 l of genomic DNA sample, 1

If there is air left over, it can create bubbles, which can puff out of the DNA of the well. Putting the tip too deeply into the well can puncture through the well causing the sample to leak out. The process of gel electrophoresis will move the DNA fragments due to the charge they contain. The size and the rate they move at are directly proportional.

l of loading dye, and 1 l of 100X SYBR stain to one 1.5 centrifuge tube
4) 5)

The loading dye will help to weigh down the DNA solution, allowing it to sink to the bottom of wells and not float. It also helps determine relative position of the DNA. The stain helps to see the DNA since DNA is white. This will move the DNA fragments allowing us to see the brightness and intensity.

Close tube and gently tap the tube Load 1 lane in the designated gel with 1 fly DNA sample 6) Use a micropipettor to load entire contents of your tube containing genomic fly DNA (12 l) into separate well in the gel. Use same procedure as Part 1 Step 10 when loading the gel 7)-12) TA will perform these steps 13) View your gel on the ultra violet trans illuminator. Make a sketch of your gel note intensity. Compare brightness with your partner.