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 14oC

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 50oC 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 37oC 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 50oC
  • 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 4oC

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 50oC
  • 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 4oC

Preparing Competent Cells Protocol

  • Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37oC and 200-300 rpm
  • Inoculate 0.25 mL of the overnight strain into 25 mL of LB
  • Shake at 37oC 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 -800oC

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

Miniprep Protocol (from QIAprep Spin Miniprep Kit)

  • Harvest cells at 5400g 10 minutes 40oC (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 -80oC 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 42oC for 45 sec
  • Incubate the cells on ice for 2 minutes
  • Under flame, add 450 µL SOC broth
  • Incubate at 37oC for 1 hour while rotating or shaking at 300rpm
  • Spread cells on appropriate antibiotic LB plates (usually 100 µL)
  • Incubate at 37oC 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