Team:US AFRL CarrollHS/Experiments

Experiments_Header_Final

Protocols

Transformations 

Bl21

  1. Thaw 50 µL vial of BL21 cells on ice
  2. Inoculate 2µL DNA into 50µL BL21 cells
    • Incubate cells on ice for 30 minutes
    • Heat shock at 42℃ for 10 seconds
    • Incubate on ice for 5 minutes
  3. Inoculate 250µL LB into vial 
  4. Plate 1x/9x

Gel protocols

(1% Agarose Electrophoresis Gel)

  1. Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask
  2. Heat in microwave for 35 seconds
  3. Swirl and heat in microwave for 25 seconds
  4. Swirl and heat in microwave for 15 seconds
  5. Swirl and heat in microwave for 7 seconds
  6. Swirl and ensure no ‘floaties’
  7. Cool outside of conical flask with water until ‘hand not’
  8. Pour into gel mold and add well comb

Gel Extraction

  1. Use ethanol-wiped scalpel to excise appropriate bands
  2. Mix and incubate at 50°C for 10 min
    • Vortex every 2 mins until gel slice is completely dissolved
  3. Place nucleospin column into 2mL collection tube
    • Add sample
    • Centrifuge at 11,000 xg for 1 minute
    • Discard throughflow and place column back in same tube
  4. Add 700 mL Buffer NT3
    • Centrifuge at 11,000 xg for 1 minute
    • Discard throughflow and place column back in same tube
  5. Repeat step 4
  6. Centrifuge at 11,000 xg for another minute
    • To remove buffer
      • Discard throughflow
      • Centrifuge at 11,00 xg for add minute
      • Place column in a clean, labelled 1.5 mL tube
  7. Add 25 mL prewarmed (50°C) Buffer NE
    • Incubate at 50°C for 5 min
    • Centrifuge at 50 xg for 1 minute
    • Centrifuge at 11,000 xg for 1 minute

Protein Gel Electrophoresis 

Sample Preparation

  1. Prepare suitable amount of sample buffer (25 µl per reaction).
  2. Determine protein concentration with NanoDrop.
  3. Calculate required dilutions needed to get 150 µg protein in 75 µl of solution.
  4. In the fume hood, add 25 µl of the sample buffer (blue) to 75 µl of protein samples (total volume of 100 µl). Centrifuge samples at 13000 rpm for 60 seconds.
  5. Incubate samples for 5 minutes in a dry heating block at 90 ◦C.
  6. Centrifuge samples at 13000 rpm for 60 sec.
  7. Store all samples at −20 ◦C

Gel Preparation (Separating Gel)

  1. Prepare the gel casts in holders. Fill the holder with water to check for leakage after 5 min. After leakage check, pour all water from gel casts and dry residual with paper towel.
  2. To prepare separation-gel, ix the reagents listed in the table below with the proper volumes to receive a gel with desired composition.
  3. Add TEMED and APS last, solidification of the gel will occur when these reagents are added.
  4. Pipette gel mixture into gel casts up to a height approximately 1 cm below the gels comb.
  5. Fill the remaining cast with isopropanol (if not available use distilled water).
  6. Allow to harden for 30-60 min.
  7. Pour all isopropanol (or water) from gel casts and dry residual with paper towel.
    • Ammonium Persulfate (APS) - 20 mg/ml
    • Acrylamide 30 % solution
    • TEMED
    • Separating Gel Buffer - 1.5 M Tris-HCl pH 8.8 with 0.4% SDS. Stored at 4 ◦C.
    • Stacking Gel Buffer - 0.5 M Tris-HCl pH 6.8 with 0.4% SDS. Stored at 4 ◦C

Gel Preparation (Stacking Gel)

  1. To prepare stacking-gel, in a tube mix the reagents listed in table 4 below with the proper volumes to receive a gel with desired composition.
  2. Add TEMED and APS last, solidification of the gel will occur when these reagents are added.
  3. Pipette gel mixture on top of separating gel to fill cast and remove all air bubbles.
  4. Fill the remaining cast with isopropanol (if not available use distilled water).
  5. Insert the comb whilst the gel is still in liquid form. The comb will form wells to load samples when the gel turns solid. 
  6. Allow to harden for 45-60 min.

Running the gel

  1. Mount the gels into the tank, remove combs and fill the inner chamber of the tank with 1x running buffer to the top. Fill a third of the volume in the outer chamber of the tank with 1x running buffer
  2. Pipette 2 µl ladder and 10 µl sample into each well.
  3. Connect power pack (red-red, black-black) and run at 100 V, 60 min or until the blue line almost reaches the bottom of the gel.
  4. Once electrophoresis has finished, remove gels from the glass plates.
  5. Carefully wash all equipment used for electrophoresis with water.

Plates 

Chloramphenicol

  1. Follow LB Agar Protocol
  2. Put in water bath 42°C
    • Cool til not burning hand
  3. Put on stirring plate
  4. Put sterilized plates in Lamenia shield
  5. Using micropipette put .5 mL of chloramphenicol into agar solution
    • 1000x dilution
  6. Pour solution into plates

Kanamycin

  1. Follow LB Agar Protocol
  2. Put in water bath 42°C
    • Cool til not burning hand
  3. Put on stirring plate
  4. Put sterilized plates in Lamenia shield
  5. Using micropipette put .5 mL of 50 ng kanamycin into agar solution
    • 1000x dilution
  6. Pour solution into plates

QIA Prep Spin Mini Prep Kit

  1. Example for 18 mL LB add
    • 18 L20 mg/ L Kanamycin (antibiotic)
    • 18 L 50 mg/ L ampicillin
  2. Aliquot 3.5 mL into labelled round bottom falcon tubes
  3. Using sterile toothpicks- pick a single colony forming unit (cfu) and inoculate tube
    • Incubate overnight at 37°C with 215 rpm shaking
  4. To 100 mL 60% glycerol stock, add 300mL overnight culture to appropriately labelled tube
    • Store at -80℃
  5. Pellet remaining cells by centrifugation in 1.5 mL tube at 13,2000 rpm for 1 min
    • Discard supernatant and repeat
  6. Resuspend pellet in Buffer P1 250 mL
    • Add 250 L Buffer, P2 and mix by inverting 10x
    • Add 350 L Buffer N3 and immediately mix by inverting 10x
  7. Centrifuge at 13, 200 rpm for 10 minute
  8. Pipette supernatant into labelled QIA prep spin column
    • Centrifuge at 13, 200 rpm for 1 minute
    • Discard Flowthrough
  9. Wash column by adding .5 mL Buffer PB *regular for low copy number plasmids*
    • Centrifuge at 13, 200 rpm for 1 minute
    • Discard flowthrough
  10. Wash by adding .75 mL Buffer PE
    • Centrifuge at 13, 200 rpm for 1 minute
    • Discard flowthrow
    • Centrifuge for an addition minute to ensure removal of residual wash buffer
  11. Place spin column in a clean, labelled 1.5 mL tube
    • Add 50 LEB to center of column & let stand for 1 minute at room temperature
    • Centrifuge at 12,000 rpm for 1 minute
    • Store at -20℃

ZYMO Pure II Plasmid Maxiprep Kit 

  1. Take 1 1.5 ml microcentrifuge tube and place 200 microliters of ZymoPURE Elution Buffer (EB)
  2. Place EB tube in a 50 C heat block
  3. Using a 50 ml pipette tip, pipette 50 ml of your 200 ml overnight into 4 Falcon tubes
  4. Centrifuge the Falcon tubes at 2,500 x g for 20 minutes to pellet the cells. Discard the supernatant.
  5. Using a 25 ml pipette tip, pipette 14 ml of ZymoPURE P1 (red) to one of the Falcon tubes and resuspend the cells.
  6. Transfer the 14 ml of your cells and P1 into another Falcon tube and resuspend. Repeat for each Falcon tube.
  7. Add 14 ml of ZymoPURE P2 (green) and immediately mix by gently inverting tube 6 times. DO NOT VORTEX. Let it sit at room temperature for 2.5 minutes. Cells will be completely lysed when the solution appears clear, purple and viscous.
  8. Add 14 ml of ZymoPURE P3 (yellow) and mix gently but thoroughly by inversion. DO NOT VORTEX. The sample will yellow when the neutralization is complete amd a yellowish precipitate will form.
  9. Ensure the plug is attached to the Luer Lock at the bottom of the ZymoPURE Syringe Filter. Place the syringe filter upright in a tube rack and load the lysate into the ZymoPURE Syringe Filter and wait 5-8 minutes for the precipitate to float to the top.
  10. Remove the Luer Lock plug from the bottom of the syringe and place the syringe into a clean 50 ml conical tube. Place the plunger in the syringe and push the solution through the ZymoPURE Syringe Filter in one continuous motion until approximately 33-35 ml of cleared lysate is recovered. Save the cleared lysate!
  11. Add 14 ml of ZymoPURE Binding Buffer to the cleared lysate from step 5 and mix thoroughly by inverting the capped tube 10 times
  12. Follow Vacuum Protocol below. Vacuum pump should be able to apply at least 400 mm Hg pressure
  13. Ensure the connections of the Zymo-Spin V-P Column Assembly are finger-tight and place onto a vacuum manifold.
  14. With the vacuum off, add the entire mixture from step 6 into the Zymo-Spin V-P Column Assembly, and then turn on the vacuum until all of the liquid has passed completely through the column.
  15. With the vacuum OFF, add 5 ml of ZymoPURE Wash 1 to the 15 ml Conical Reservoir. Turn on the vacuum until all of the liquid has passes completely through the column.
  16. With the vacuum OFF, add 5 ml of ZymoPURE Wash 2 to the 15 ml Conical Reservoir. Turn on the vacuum until all of the liquid has passes completely through the column.
  17. Repeat Step 14.
  18. Remove and discard the 15 ml Conical Reservoir and place the Zymo-Spin V-P Column in a Collection Tube. Centrifuge at 13,400 x g for 1 minute, in a microcentrifuge, to remove any residual wash buffer.
  19. Transfer the column into a clean 1.5 ml microcentrifuge tube and add 200 microliters of the heated EB directly into the column matrix. Wait 10 minutes and then centrifuge at 13,400 x g for 1 minute.
  20. Nanodrop to measure DNA concentration of your maxiprep. 



QuikChange Site-Directed Mutagenesis

  1. Set up the series of various concentrations of dsDNA template (5, 10, 20, and 50 ng of dsDNA)

    • pY71-sfGFP ≈ 200 ng/μL

      5 ng/μL = 1:40 ( 39 μL of ddH₂O + 1 μL of pY71-sfGFP)

      10 ng/μL = 1:20 (19 μL of ddH₂O + 1 μL of pY71-sfGFP)

      20 ng/μL = 1:10 (9 μL of ddH₂O + 1 μL of pY71-sfGFP)

      50 ng/μL = 1:4 (3 μL of ddH₂O + 1 μL of pY71-sfGFP)

  2. Did a 10x dilution of the primer #1 T7-mutF and primer #2 T7-mutR
  3. Set up a master mix for 6 reactions ( 4 different concentrations of dsDNA, 1 blank, and 1 extra)

    • 30 μL of 5x iProof Buffer

      3 μL of T7-mutF

      3 μL of T7-mutR

      3 μL of DNTP

      105 μL of ddH₂O

      3 μL of iProof DNA polymerase

  4. Place 24.5 μL of the master mix into 5 different labeled PCR tubes(5, 10, 20, 50 ng of pY71-sfGFP and a blank)

    • Add 0.5 μL of the corresponding DNA to each DNA tube

      Add 0.5 μL of ddH₂O to the blank labeled tube

  5. PCR Protocol

Preheat lid to 98℃

Segment

Cycles

Temperature (℃)

Time

1

1

95

30 seconds

2

12

95

30 seconds

55

1 minute

68

1 min/kb of plasmid length, 2.5 min for pY71-sfGFP

3

1

4

2 minutes

MAKE SURE TO REPEAT SEGMENT 2 FOR 12 CYCLES

6. After PCR reaction is finished, add 0.5 μL of Dpn I restriction enzyme.

Gently mix by pipetting the solution up and down several times. Spin down the reactions in a microcentrifuge for 1 minute

7. Immediately incubate reactions at 37 ℃ for 1 hour to digest

Can be done in either an incubator or a thermocycler

8. After incubation, do 2 plasmid transformations into DH5α cells for each PCR tube, 1 with 1 μL of DNA and the other with 5 uL of DNA

100 μL of DH5α cells

  • Supplement the change of DNA used with ddH₂O:

    • If adding 1 μL of DNA

      Add 20 μL 5x KCM and 79 μL of ddH₂O,

      If adding 5 μL of DNA

      Add 20 μL 5X KCM and 75 μL of ddH₂O

  • Ice for 30 minutes
  • Heatshock for 45 seconds at 42 ℃
  • Ice again for 2 minutes
  • Add 900 μL of LB Broth
  • Shake and incubate for an hour at 37 ℃
  • Spin tubes down at 800 x g for 5 minutes
  • Remove 1000 μL, and resuspend pellet
  • Plate the whole tube



Gibson Assembly® Protocol (E5510)

Set up the following reaction on ice:

  1. Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.

- NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool,NEBioCalculator.

- The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.

2-3 Fragments Assembly

4-6 Fragments Assembly

Positive Control **

Concentration Range of DNA fragments

02 - .5 pmols*

.2 - 1.0 pmols*

0 pmols

Total Volume of Fragments (μl)

10

Gibson Assembly Master Mix (2x) (μl)

10

10

10

Deionized Water (μl)

#VALUE!

#VALUE!

0

Total Volume (μl) ***

20

20

20

*Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.

** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.

*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.

  1. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.

Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details seeFAQ section).

*Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments

Timer: 15 m

  1. Store samples on ice or at –20°C for subsequent transformation.
  2. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following thechemcial transformation protocol orelectro competent cells transformation protocol



Ligation Protocol WITH T4 DNA Ligase (M0202)

Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. UseNEB calculator to calculate molar ratios.

  1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.
    • Tip: Aliquot the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.
  2. Set up the following reaction in a microcentrifuge tube on ice:

Component

Volume (μl)

10X T4 DNA Ligase Buffer

2

Vector DNA: 50 ng (0.020 pmol)

Insert DNA: 37.5 ng (0.060 pmol)

Nuclease-free water

17

T4 DNA Ligase

1

Total

20

3. Gently mix the reaction by pipetting up and down and microfuge briefly.

4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.

5. Heat inactivate at 65 degrees C for 10 minutes.

Timer: 10 m

6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Use 25 uL DH5α cells, and add 2 uL of reaction mixture.

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1X TAE Buffer

Formula: M₁ V₁ = M₂ V₂

(50X) V₁ = 1X (1000mL) V₁ = 20mL50X TAE

  1. Pour 20mL of 50X TAE into a graduated cylinder, then pour into 1L bottle.
  2. Take 1L bottle with 20mL of 50X TAE and fill to 1000mL line (not labelled, indented) with 18mo water.

    • Actually filling bottle with 980 mL of 18mo water. (20 + 980 = 1000mL)

  3. Ethidium bromide: (10mg / mL) x (10³ mg / mg) = 1 x 10⁴ μg / mL (concentration in bottle)

    • (1 x 10⁴ μg / mL) V₁ = 0.05 μg / mL (1000 mL) V₁ = 50μL

  4. Pipette 50 μL of etBr with 100 μL pipette and release into 1L bottle containing 20 mL of 50X TAE and 980 mL of 18 mo of water
  5. Swirl bottle.
  6. Cover completely in foil (1L bottle).
  7. Label “1X TAE 0.5 μg / mL etBr” on cap with tape.

Glycerol Stock

  1. In a 1.5 mL microcentrifuge tube, pipette 100 mL of the 60% glycerol
  2. In the same tube, pipette 300 mL of the overnight of your desired plasmid
  3. Store tube in a -80 °C freezer

LB Agar

  1. Gather material and place them into 1 empty 500 mL Erlenmeyer Flask Make sure to put stir bar in

    • Needed to make into homogenous gel Needs to be sterile

  2. Put into Autoclave
    • Lid should not be too tight


LB Broth

  1. Place materials in a flask and create a homogenous solution
  2. Place flask with material in the autoclave Lid should not be on too tight
  3. Lid should be completely tighten after it is finished and taken out of the autoclave

Overnights

  1. Fill your Falcon tube with 3 μL of LB Broth and 3 mL of 25 mg/mL Chloramphenicol
  2. Using the toothpick pick a single colony from your plate
  3. Place toothpick with colony inside of the Falcon tube
  4. Repeat Steps 1-3 as needed
  5. Close your Falcon tube(s) and place it/them in the incubator overnight at 37o C