Team:BostonU/Protocols

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PROTOCOLS

Below is an interactive list of the laboratory techniques employed by the team. Click to navigate to that protocol.

KOD PCR, Colony PCR, Overlap Extention PCR, Making a 1% Agarose Gel, Agarose Gel Electrophoresis, Cell Stock, Miniprep, Gel Extraction, PCR Cleanup, Digestion, Ligation, Transformation, Liquid Cultures, Test Cuts, Gibson, Recombination Reaction, Cell-Free Reaction

 

KOD PCR

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

Number of Cycles	From 20 - 40
Annealing Temperature	Set temperature to the lowest primer melt temperature
Extension Time	If target size is: < 500 bp run 10 sec/kb 500-1000 bp run 15 sec/kb 1000-3000 bp run 20 sec/kb < 3000 bp run 20 sec/kb

 

Colony PCR

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

Number of Cycles	From 20 - 40
Annealing Temperature	Set temperature to the lowest primer melt temperature
Extension Time	If target size is: < 500 bp run 10 sec/kb 500-1000 bp run 15 sec/kb 1000-3000 bp run 20 sec/kb < 3000 bp run 20 sec/kb

 

Overlap Extension PCR

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

Number of Cycles	From 20 - 40
Annealing Temperature	Set temperature to the lowest primer melt temperature
Extension Time	If target size is: < 500 bp run 10 sec/kb 500-1000 bp run 15 sec/kb 1000-3000 bp run 20 sec/kb < 3000 bp run 20 sec/kb

 

Making a 1% Agarose Gel

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

 

Agarose Gel Electrophoresis

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

 

Cell Stock

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℃

 

Miniprep

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℃

 

Gel Extraction

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℃

 

PCR Cleanup

Materials

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

Procedure

1. Add 10-100ul of PCR product into 1.7ml microcentrifuge tube
2. Add 500ul of PX buffer to microcentrifuge tube
3. Transfer contents to spin columns that are place on the vacuum manifold. Turn on manifold
4. Pipette 500 uL of WN buffer into each column
5. Pipette 500 uL of WS buffer into each column
6. Turn of vacuum manifold and place spin columns in collection tubes
7. Centrifuge for 3 minutes at MAX speed and discard collection tubes. Place the spin column in a new microcentrifuge tube
8. 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.
9. Centrifuge for 60 seconds at MAX speed
10. Nanodrop DNA for concentration and store in at -20℃

 

Digestion

Materials

• Restriction Enzymes (keep on ice or ice block)
• Restriction Enzyme Buffer
• DNA
• Deionized water
• PCR Tubes

Procedure

1. Add 4ug of plasmid DNA or 2ug of linear DNA to PCR tube
2. Total amount of enzymes added should be 4ul
3. Add 5ul of compatible buffer for restriction enzymes
4. Add deionized water to a total volume of 50ul
5. Place in thermocycler
6. If adding CIP- add 1ul of CIP to the sample in the final 20 minutes while the sample remains in the thermocycler

 

Ligation

Materials

• T4 DNA Ligase (keep on ice or ice block)
• T4 DNA Ligase Buffer
• DNA
• Deionized Water
• PCR Tubes

Procedure

1. Add equimolar concentrations of DNA fragments to PCR tube
2. Add equimolar concentrations of DNA fragments to PCR tube
3. Add 1ul of T4 DNA ligase
4. Add deionized water to a final volume of 20ul
5. Leave on bench for 20 minutes
6. Heat inactivate by placing sample in 70℃ bead bath for 1 minute

 

Transformation

Materials

• KCM
• Top 10 Competent Cells
• Deionized Water
• PCR Tubes

Procedure

1. Add 10ul of KCM to sample
2. Add deionized water to 50ul
3. Transfer contents of sample to Top Ten Cells
4. Place in thermocycler when the block reaches 4℃
5. Once finished in thermocycler spread the entire sample onto plates

 

Liquid Cultures

Materials

• 12ml Culture Tubes
• Liquid Media

Procedure

1. Add 4ml of liquid media to culture tube
2. Pick colony and eject tip into culture tube
3. Incubate in a 37°C shaking incubator for 16-18 hours

 

Test Cuts

Materials

• Restriction Enzymes
• Restriction Enzyme Buffer
• DNA
• Deionized Water
• Microcentrifuge Tubes
• PCR Tubes

Procedure

1. Make a master mix with the following volumes in a microcentrifuge tube: 1ul of Buffer, 0.25ul of each Restriction Enzyme, 6.5ul of Deionized Water
2. Add 2ul of DNA to 8ul of Master Mix in a PCR tube
3. Incubate reaction at 37°C for at least 15-30 minutes
4. Run on agarose gel

 

Gibson

Materials

• DNA insert
• DNA backbone
• Deionized Water
• Gibson Master Mix
• PCR tubes

Procedure

1. Add equimolar concentrations of DNA insert and DNA backbone to PCR tube
2. Add deionized water to a total volume of 10ul
3. Add 10ul of Gibson Master Mix
4. Place in thermocycler

 

Recombination Reaction

Materials

• 250ng of DNA
• 10X of Recombinase Buffer
• Recombinase
• Deionized Water
• PCR Tubes

Procedure

1. Add 250ng of DNA
2. Add 5ul of 10X Recombinase Buffer
3. Add 1ul of Recombinase
4. Add deionized water to total volume of 50ul
5. Incubate reaction at 37°C for 30 minutes
6. Hit Shock at 70°C for 10 minutes

 

Cell-Free Reaction

Materials

• Cell Free Crude Cell Extract Master Mix
• Autoclaved Water
• DNA
• 384 Well Plates
• PCR Tubes
• Plate Reader
• Ice

Procedure

1. Add 4nM of DNA to each PCR tube
2. Add 16.5ul of Cell Free Crude Cell Extract Master Mix to PCR tube
3. Add autoclaved water to total volume of 20ul
4. Centrifuge PCR tubes
5. Transfer reactions from PCR tubes to a 384 Well Plates
6. Take initial fluorescence reading using a plate reader with wavelengths at 485nm and 525nm
7. Incubate plate at 30°C
8. Measure fluorescence every hour, we found that fluorescence levels out at 6 hours