Protocols

Ligation Protocol

• Determine insert to vector ratios
• Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)
• In a PCR tube add the following:
  • 50ng of vector
  • Amount of insert based on ratios (calculated in second step)
  • 2uL of buffer
  • 2uL of DNA ligase
  • Amount of water to bring total volume to 20uL
• Incubate overnight at 14^oC

Note: We used T4 DNA ligase and buffer from NEB

Gel Purification Protocol (from QIAquick Gel Extraction Kit)

• Excise DNA fragment from the agarose gel with a clean, sharp scalpel
• Weigh the gel slice in a microcentrifuge tube.
• Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)
• Incubate at 50^oC for 10 min (until the gel slice has completely dissolved)
• After the gel slice has dissolved completely, check that the color of the mixture is yellow
• Apply the sample to a QIAquick column, and centrifuge for 1 min
  • Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
• Discard flow-through and place QIAquick column back in the same collection tube
• To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.
• Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm
• Place QIAquick column into a clean 1.5 mL microcentrifuge tube
• To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.

Gel Electrophoresis Protocol

• Making a 1% agarose gel
  • 100mL 1X TBE buffer
  • 1g agarose
  • microwave until agarose dissolves
  • let mixture cool
  • when cool add 8-10uL ethidium bromide
  • stir gently, let cool
  • pour into plate with comb already in place
  • let harden
• Using the gel
  • Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)
  • Load 2uL of DNA ladder into the gel
  • Load DNA into the gel
  • Run at 130V for 30min-1hr

Digestion Protocol

• Using a microcentrifuge tube add the following:
  • ~3000-5000 ng of DNA
  • 10uL Buffer 4
  • 10uL BSA
  • 5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)
  • Amount of H₂O needed to make final volume 100uL
• Incubate at 37^oC for 1hr and 30min

Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.

Preparing LB+Appropriate Antibiotic Protocol

• 200 mL LB broth
• Autoclave
  • Put control thermometer in H₂O (from the sink)
  • Select vented container mode (Do Not Change Program)
• Let cool to 50^oC
• Add antibiotic (50-100 ug/mL) (10 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H₂O
  • Add to LB mixture when cool enough
• Store at 4^oC

Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)

• 300 mL DI H2O + 11 g LB agar
• Autoclave
  • Put control thermometer in H₂O (from the sink)
  • Select vented container mode (Do Not Change Program)
• Mix well after autoclaving; let cool to 50^oC
• Add antibiotic (50 to 100 µg/mL) (15 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H₂O
  • Add to LB mixture when cool enough
• Plate
  • Under flame open lids of all plates
  • Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring
  • Let sit under flames until gel solidifies
  • Replace lids on plates
• Store upside down at 4^oC

Preparing Competent Cells Protocol

• Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37^oC and 200-300 rpm
• Inoculate 0.25 mL of the overnight strain into 25 mL of LB
• Shake at 37^oC until the OD650 is 0.6-0.7
• Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl₂
• Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl₂
• Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl₂
• Leave on ice for 30 minutes
• For long term storage, use 0.1M CaCl₂ in 15% glycerol at step 6 and store cells at -800^oC

Note: Harvest cells at 5000 rpm for 10 minutes at 4^oC

Miniprep Protocol (from QIAprep Spin Miniprep Kit)

• Harvest cells at 5400g 10 minutes 40^oC (possibly program 1)
• Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube
• Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times
• Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
• Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge
• Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting
• Centrifuge for 30-60 seconds. Discard the flow-through
• Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds
• Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer
• To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.

Preparing Glycerol Stock Protocol

• Add 150 µL of 50% glycerol to 350 µL of cells
• Place in -80^oC freezer

Transformation Protocol

• With a pipette tip, punch a hole through the foil cover of the DNA plate
• Add 10 µL of DI water
• Thaw competent cells on ice
• Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes
• Incubate the cells on ice for 30 minutes
• Heat shock the cells at 42^oC for 45 sec
• Incubate the cells on ice for 2 minutes
• Under flame, add 450 µL SOC broth
• Incubate at 37^oC for 1 hour while rotating or shaking at 300rpm
• Spread cells on appropriate antibiotic LB plates (usually 100 µL)
• Incubate at 37^oC for 18-24 hours
• Take a colony, put in 3 mL of LB + appropriate antibiotic
• Use resulting culture to miniprep DNA and make your own glycerol stock