Difference between revisions of "Team:BostonU/Protocols"

Materials

• 5% DMSO
• 2x KOD Hot Start Master Mix
• PCR Tubes
• Forward Primers(s) [10 uM]
• Reverse Primer(s) [10 uM]
• DNA Template
• Deionized water

Procedure

1. Add 18.5 uL of deionized water to PCR tube
2. Add 1.5 µl of both the requisite Forward and Reverse Primers to PCR tube
3. Add 1 ul of DNA template
4. Add 27.5 µl of KOD Hot start Master Mix to PCR tube
5. Centrifuge for 10 seconds to remove air bubbles
6. Place in Thermocycler

Thermocycler Conditions

Materials

• 5% DMSO
• 2x KOD Hot Start Master Mix
• PCR Tubes
• Forward Primers(s) [10 uM]
• Reverse Primer(s) [10 uM]
• DNA Template
• Deionized water

Procedure

1. Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube
2. Add 17.5 µl of deionized water to another PCR tube
3. Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube
4. Add 2 ul of water containing cells with DNA template sample
5. Add 27.5 µl of KOD Hot Start Master Mix
6. Centrifuge for 10 seconds to remove air bubbles
7. Place in Thermocycler

Thermocycler Conditions

Materials

• 5% DMSO
• 2x KOD Hot Start Master Mix
• PCR Tubes
• Forward Primers(s) [10 uM]
• Reverse Primer(s) [10 uM]
• DNA Template
• Deionized water

Procedure

1. Add equimolar concentrations of each DNA sample to a PCR tube
2. Add 1.5 uL of each Forward and Reverse Primer
3. Add 27.5 uL of KOD Hot Start Master Mix
4. Add deionized water until the total volume is 50 uL
5. Place in Themocycler

Thermocycler Conditions

Materials

• Agarose
• 1x TAE Buffer
• Ethidium Bromide
• 250 mL Flask

Procedure

1. Add 0.6 g of agarose to 250 mL flask
2. Add 60 mL of 1x TAE buffer and mix by swirling the flask
3. Microwave mixture for 60 seconds
4. Add 3 uL of Ethidium Bromide and mix gently
5. Pour into gel tray with the desired comb size and wait 30 minutes for it to solidify

Materials

• Agarose Gel
• 6x Loading Dye
• 2 log DNA ladder
• Gel Box
• 1x TAE Buffer

Procedure

1. Add 6x loading dye to each sample
2. Place agarose gel into gel box
3. Fill gel box with 1x TAE buffer to fill line
4. Load 2 log ladder and samples into wells
5. Run gel at 135 V for 30-40 minutes

Materials

• Liquid Cell Culture
• 50% Glycerol
• Cryofreezer Tube

Procedure

1. Add 600 ul of glycerol into cryotube
2. Add 600 ul of culture to cryotube
3. Mix gently by pipetting up and down
4. Freeze and store at -80℃

Materials

• 1.7 mL Microcentrifuge Tubes
• Spin Columns
• Spin Tubes
• MX1 Buffer
• MX2 Buffer
• MX3 Buffer
• WN Buffer
• WS Buffer
• Elution Buffer
• Centrifuge
• Vacuum Manifold
• 55℃ Bead Bath

Procedure

1. Prepare the Elution Buffer by placing it in the 55℃ bead bath
2. Transfer the sample to a microcentrifuge tube and spin for 2 minutes at 13,000 rpm
3. Pour off the supernatant from the pellet
4. Pipette 200 uL of MX1 into the samples and vortex to resuspend
5. Pipette 250 uL of MX2 into the samples and invert the tube ~5 times
6. Pipette 350 uL of MX3 into the samples and invert the tube ~5 times
7. Centrifuge at MAX speed for 5 minutes
8. Pour the supernatant off the microcentrifuge tubes into spin columns and turn on the vacuum manifold
9. Pipette 500 uL of WN buffer into each column
10. Pipette 700 uL of WS buffer into each column
11. Turn of vacuum manifold and place spin columns in collection tubes
12. Centrifuge for 3 minutes at MAX speed and discard collection tubes. Place the spin column in a new microcentrifuge tube
13. Pipette 50 uL of Elution Buffer right above the membrane of the column without touching the membrane with the pipette tip. Let the column sit for 2 minutes
14. Centrifuge for 60 seconds at MAX speed
15. Nanodrop DNA for concentration and store in at -20℃

Materials

• 1.7ml Microcentrifuge Tubes
• Spin Columns
• Spin Tubes
• GEX Buffer
• WN Buffer
• WS Buffer
• Centrifuge
• Vacuum Manifold
• 55℃ Bead Bath

Procedure

1. Prepare the Elution Buffer by placing it in the 55℃ bead bath
2. Place excised gel fragment in a microcentrifuge tube and add 700 uL of GEX buffer
3. Place tubes in the 55℃ bead bath and wait for the gel fragment to fully dissolve
4. Transfer contents to spin columns that are place on the vacuum manifold. Turn on manifold
5. Pipette 500 uL of WN buffer into each column
6. Pipette 500 uL of WS buffer into each column
7. Turn of vacuum manifold and place spin columns in collection tubes
8. Centrifuge for 3 minutes at MAX speed and discard collection tubes. Place the spin column in a new microcentrifuge tube
9. Pipette 30 uL of Elution Buffer right above the membrane of the column without touching the membrane with the pipette tip. Let the column sit for 2 minutes.
10. Centrifuge for 60 seconds at MAX speed
11. Nanodrop DNA for concentration and store in at -20℃

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