materials
Gel box
Aragose gel
TAE buffer concentrate, 40X
Tube rack for 1.7 mL
Reaction tubes, 1.7 mL
Permanent lab marker pens
DNA samples
Yeast DNA, 50 yg/uL + loading dye
pBR322, 50 ug/uL + loading dye
Lambda DNA, 50 ug/uL + loading dye
Other DNA samples + loading dye
Gel loading dye, 6x
Micropipet, P-10
Micropipet, P-100
Micropipet tips. P-10
Micropipet tips, P-100
Microcentrifuge
Lambda/Hind III, 50 ug/uL + dye
Power Supply
Ethidium Bromide, 0.5 ug/mL
Gel photo imaging system
Paper, thermal
Printer, thermal
Gloves, large
Glasses
Weigh boat, 5.5" X 5.5"
EDTA, disodium salt
Bottle, 125 mL
Graduated cylinder, 100 mL
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
Glass rods
Filtering flasks, 250 mL, 0.2 um
Vacuum pump and "trap" jar
TAE concentrate, 40x
Beakers, 600 mL
Agarose
Balance, tabletop miligram
Weigh Boat 3.5" X 3.5"
Lab scoops
Media bottle, 250 mL
Permanent Lab Marker Pens
Microwave
Hot hands protectors
Glasses
Gel box, Horizontal, for agrose gels
Beakers, 50 mL
Water bath, 65 celcius
Beakers, 50 mL
DNA, salmon testes
Pipet, 2 mL
Pipet pump, blue
Micropipet, p-1000
Micropipet tips
Ethanol, 95%
Glass rods
Tubes, 15 mL capped
Tube racks for 15 mL tubes
Lab markers
Plastic beaker, 1L tripour
Balance, analytical
Balance, tabletop miligram
Weigh paper, 7.6 X 7.6 cm
Weigh boat, 3.5" X 3.5"
Lab scoops
Sodium Chloride
Tubes, 15 mL, capped
Tube racks for 15 mL tubes
TRIS
Aragose gel
TAE buffer concentrate, 40X
Tube rack for 1.7 mL
Reaction tubes, 1.7 mL
Permanent lab marker pens
DNA samples
Yeast DNA, 50 yg/uL + loading dye
pBR322, 50 ug/uL + loading dye
Lambda DNA, 50 ug/uL + loading dye
Other DNA samples + loading dye
Gel loading dye, 6x
Micropipet, P-10
Micropipet, P-100
Micropipet tips. P-10
Micropipet tips, P-100
Microcentrifuge
Lambda/Hind III, 50 ug/uL + dye
Power Supply
Ethidium Bromide, 0.5 ug/mL
Gel photo imaging system
Paper, thermal
Printer, thermal
Gloves, large
Glasses
Weigh boat, 5.5" X 5.5"
EDTA, disodium salt
Bottle, 125 mL
Graduated cylinder, 100 mL
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
Glass rods
Filtering flasks, 250 mL, 0.2 um
Vacuum pump and "trap" jar
TAE concentrate, 40x
Beakers, 600 mL
Agarose
Balance, tabletop miligram
Weigh Boat 3.5" X 3.5"
Lab scoops
Media bottle, 250 mL
Permanent Lab Marker Pens
Microwave
Hot hands protectors
Glasses
Gel box, Horizontal, for agrose gels
Beakers, 50 mL
Water bath, 65 celcius
Beakers, 50 mL
DNA, salmon testes
Pipet, 2 mL
Pipet pump, blue
Micropipet, p-1000
Micropipet tips
Ethanol, 95%
Glass rods
Tubes, 15 mL capped
Tube racks for 15 mL tubes
Lab markers
Plastic beaker, 1L tripour
Balance, analytical
Balance, tabletop miligram
Weigh paper, 7.6 X 7.6 cm
Weigh boat, 3.5" X 3.5"
Lab scoops
Sodium Chloride
Tubes, 15 mL, capped
Tube racks for 15 mL tubes
TRIS
purpose
Procedure: lab 4a nad 4b
1: Molarity calculations
Solution One: NaCl: 5M x 0.010 L x 58.44 g/mole = 2.92 grams
Solution Two: TRIS: 0.01 M x 0.1 L x 372.24 g/mole = 0.158 grams
Solution Three: 0.001 M x 0.1 L x 372.24 g/mole = 0.037 grams
2: Create TE Solution by combining Solutions Two and Three
3: Dilute DNA with TE solution in a flask.
4: Add 2.92 grams of NaCl.
5: Add 4 mL of alcohol by trickling it down the side of the flask.
6: Spool DNA using glass rod
7: Put the spooled DNA into a new tube and add 2 mL of fresh TE solution
Procedure: lab 4i
1: 500 mL of 1x tae from 40x stock
1x tae Buffer- (40x)(V) = (500 mL)(1x) V1 = 12.5 mL
2: 50 mL .8% agarose
Agaorse- (0.008)(50 mL) = 0.4 grams
3: Add 0.8% agarose to 100 mL 1X tae buffer solution.
2: Weigh out the required mass of powdered agarose in a weigh boat. Add it to a 250 mL media bottle.
3: Measure out enough tae buffer to prepare a total of 100 mL of agarose and buffer mixed together. Swirl to mix.
4: Cap the media bottle and swirl the flask to suspend the agarose in the buffer.
5: To dissolve the agarose, microwave for 4 minutes at 50% power. Wait for the solution to boil.
6: Place the hot dissolved agarose solution on a fireproof lab tabletop and let cool to 65 degrees Celsius before pouring into a gel tray.
7: Place a six-well comb into the notches at the end of the gel tray. This will create the necessary wells for the next experiment.
8: Place the gel (on the gel tray) into a gel box.
9: Pour 1X tae buffer into the gel box and completely submerge the gel.
10: Gently pull the comb out of the gel and make sure the wells are not broken or cracked.
Procedure: lab 4j
1: Prepare the gel and gel box for loading.
2: Carefully secure gates of the gel tray. Make sure the 1X tae buffer covers the gel by at least 1 centimeter.
3: Obtain 1.7 mL tubes.
4: Add 20 micro liters of salmon sperm DNA and add 4 micro liters of 6X DNA loading dye. Spin the sample in a minicentrifuge.
5: Load the salmon sperm DNA sample into the wells using a micro pipette.
6: Connect the electrodes of the gel box to the power supply and run the gel at 110 Volts for 45 minutes.
7: Run until you can see the front loading dye halfway down the gel.
8: Cover the gel with EtBr (ethinium bromide) and stain the gel for 20 minutes. Then pour off the EtBr, cover with disstilled water, and observe the gel on a UV light box.
9: Analyze the contents of each well, size of the standards, the samples, and the DNA bands.
1: Molarity calculations
Solution One: NaCl: 5M x 0.010 L x 58.44 g/mole = 2.92 grams
Solution Two: TRIS: 0.01 M x 0.1 L x 372.24 g/mole = 0.158 grams
Solution Three: 0.001 M x 0.1 L x 372.24 g/mole = 0.037 grams
2: Create TE Solution by combining Solutions Two and Three
3: Dilute DNA with TE solution in a flask.
4: Add 2.92 grams of NaCl.
5: Add 4 mL of alcohol by trickling it down the side of the flask.
6: Spool DNA using glass rod
7: Put the spooled DNA into a new tube and add 2 mL of fresh TE solution
Procedure: lab 4i
1: 500 mL of 1x tae from 40x stock
1x tae Buffer- (40x)(V) = (500 mL)(1x) V1 = 12.5 mL
2: 50 mL .8% agarose
Agaorse- (0.008)(50 mL) = 0.4 grams
3: Add 0.8% agarose to 100 mL 1X tae buffer solution.
2: Weigh out the required mass of powdered agarose in a weigh boat. Add it to a 250 mL media bottle.
3: Measure out enough tae buffer to prepare a total of 100 mL of agarose and buffer mixed together. Swirl to mix.
4: Cap the media bottle and swirl the flask to suspend the agarose in the buffer.
5: To dissolve the agarose, microwave for 4 minutes at 50% power. Wait for the solution to boil.
6: Place the hot dissolved agarose solution on a fireproof lab tabletop and let cool to 65 degrees Celsius before pouring into a gel tray.
7: Place a six-well comb into the notches at the end of the gel tray. This will create the necessary wells for the next experiment.
8: Place the gel (on the gel tray) into a gel box.
9: Pour 1X tae buffer into the gel box and completely submerge the gel.
10: Gently pull the comb out of the gel and make sure the wells are not broken or cracked.
Procedure: lab 4j
1: Prepare the gel and gel box for loading.
2: Carefully secure gates of the gel tray. Make sure the 1X tae buffer covers the gel by at least 1 centimeter.
3: Obtain 1.7 mL tubes.
4: Add 20 micro liters of salmon sperm DNA and add 4 micro liters of 6X DNA loading dye. Spin the sample in a minicentrifuge.
5: Load the salmon sperm DNA sample into the wells using a micro pipette.
6: Connect the electrodes of the gel box to the power supply and run the gel at 110 Volts for 45 minutes.
7: Run until you can see the front loading dye halfway down the gel.
8: Cover the gel with EtBr (ethinium bromide) and stain the gel for 20 minutes. Then pour off the EtBr, cover with disstilled water, and observe the gel on a UV light box.
9: Analyze the contents of each well, size of the standards, the samples, and the DNA bands.