Team:SVA-NYC/Laboratory Protocols

SVA-NYC

Experiments & Protocols

Laboratory Protocols

E-Coli Transformation

Transformation

1. Thaw competent cells on ice.
2. Add DNA to competent cell PCR tubes (3uL from BioBrick suspensions or 20 uL from ligations).
3. Vortex to homogenize.
4. Place in ice for 30 minutes.
5. Heat shock at 42 C for 90 seconds.
6. Place back in ice for 2 minutes to recover.
7. Add 100 uL of recovery broth and resuspend.
8. Incubate at 37 C while shaking at 300 rpm.
9. Take 2 agar plates, pipet 100 uL onto each and streak with inoculation loops.
10. Incubate overnight at 37 C.

Mini­Prep

1. Pour 6 mL cell broth into 15 mL tubes.
2. Centrifuge at 3500 rpm for 5 minutes.
3. Decant completely and discard supernatant.
4. Add 250 uL Buffer 1 to pellet.
5. Vortex to resuspend.
6. Place 250 uL Buffer 2 into a new 1.5 mL tube.
7. Take 250 uL of resuspended cells+Buffer 1 and pipet into the tube with Buffer 2.
8. Mix by inverting ten times.
9. Quickly open and add 350 uL of Buffer 3.
10. Mix by inverting twelve times.
11. Place in freezer for 10 minutes.
12. Centrifuge at max speed for 10 minutes.
13. Transfer supernatant to silica DNA bind column.
14. Incubate at room temperature for 1 minute.
15. Spin at max speed for 1 minute.
16. Remove ‘flow through’ and reassemble column.
17. Pipet 700 uL onto the silica disc at top of column.
18. Quickly spin at max speed for 1 minute.
19. Discard ‘flow through’ and spin at max speed for 1 minute again.
20. Transfer top of silica column to a new 1.5 mL tube.
21. Dispense 200 uL of 60 C diH2O onto center of silica disc.
22. Incubate at room temperature for 1 minute.
23. Spin at max speed for 1 minute.
24. Discard silica top, label tube and place in freezer.

Download E-Coli Transformation Protocol

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DNA Restriction and Ligation

DNA Restriction

1. Pre­heat PCR machine to 37 C.
2. Fill wells of PCR machine with H2O.
3. Thaw cutsmart buffer on ice.
4. Map out desired cuts on a plasmid map with measurements.
5. On ice, pipet 16 uL plasmid DNA, 2 uL cutsmart buffer, 1 uL restriction enzyme A, 1 uL restriction enzyme B (or 17 uL plasmid if only one cut­site) into a .5 mL PCR tube.
6. Repeat for the secondary gene of interest (one backbone + one gene of interest).
7. Vortex both tubes.
8. Incubate all reactions at 37 C for 2 hours in the PCR machine.
9. With ~30 minutes left, prepare a gel electrophoresis gel.
10. Run samples on gel electrophoresis with ladder (keep at least one well between different DNA).
11. Isolate and purify gel bands.
12. Freeze or use for ligation.

DNA Ligation

1. Thaw T4­Ligase buffer on ice.
2. While on ice, dispense cut 17.5 uL backbone DNA, 17.5 uL cut gene of interest, 4 uL T4­Ligase buffer, and 1 uL DNA ligase into a .5 mL PCR tube.
3. Vortex contents and label tubes.
4. Incubate at 16 C overnight (minimum of 30 minutes should suffice with minimal efficiency).
5. Proceed to transformation into bacterial chassis.

Download DNA Restriction and Ligation Protocol

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Culturing Competent Cells: (3 Day Process)

Day One

1. Take the desired bacterial strain and streak on a plate
2. Incubate at 37 C overnight

Day Two

1. Use 15mL centrifuge tube to measure out ~15mL of LB broth.
2. Add desired antibiotic in a 1:1000 ratio (~15uL).
3. Move 15mL mixture (LB+antibiotic) to a 125 mL or 250 mL erlenmeyer flask.
4. Isolate one colony using a sterile toothpick and drop into flask (ensuring the colony touches the LB, not glass).
5. Incubate at 37 C while shaking at 250 rpm overnight.

Day Three

1. Prechill 20 PCR tubes, 1 mL LB broth, and TSS Buffer.
2. Put 125 mL of LB+appropriate antibiotics into a 250 mL erlenmeyer flask.
3. Add 1 mL of overnight cell broth into the 125 mL of LB broth.
4. Incubate at 37 C while shaking at 250 rpm for 3­6 hours.
5. After 3 hours, remove 2 mL of cell broth every 30 minutes and run through a spectrophotometer (with LB as the blank).
6. Continue to do so until the optical density has reached .5 .
7. At optical density of .5, distribute broth across eight 15 mL centrifuge tubes.
8. Centrifuge at max (3500 rpm) in blood centrifuge for 10 minutes.
9. Place tubes on ice for 15 minutes.
10. While on ice, decant entire supernatant and discard.
11. Pipet 1 mL of prechilled LB broth into one of the 8 centrifuge tubes.
12. While on ice, resuspend pellet with pipet.
13. Transfer entire resuspended contents into next 15 mL tube.
14. Repeat until the contents of all eight tubes are only in one tube (all eight pellets suspended in 1 mL of LB).
15. Measure total volume by pipetting back into an empty 15 mL tube in known increments.
16. While on ice, add equal volume of chilled TSS buffer and resuspend.
17. Pipet 100 uL of cells+TSS buffer into each of the 20 PCR tubes.
18. Cap tubes, label and utilize/store in the freezer.

Download Culturing Competent Cells Protocol

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