Team:Victoria Wellington/Protocols

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Protocols
Preparation of electrocompetent cells:
from 'NEB - making your own electrocompetent cells'
  1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37°C and 250 rpm.
  2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks.
  3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
  4. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
  5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.
  6. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
  7. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
  8. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. If necessary, adjust the final volume of cells so that the OD600 is within the range 210-270.
  9. At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer.
Electroporation protocol:
from 'NEB - making your own electrocompetent cells'
  1. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
  2. Place recovery SOC in 37°C water bath.
  3. Pre-warm LB-antibiotic plates at 37°C.
  4. Thaw cells on ice for 10 min or use freshly made cells.
  5. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
  6. Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
  7. Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
  8. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
  9. Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
  10. Rotate in the 37°C incubator for 1 h.
  11. Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:
  12. Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
  13. Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
  14. Incubate overnight at 37°C.
HiFi Gibson Assembly protocol:
from 'NEB - NEBuilder HiFi DNA Assembly reaction'
Using the recommended protocol for 4-6 gene fragments
  1. Set up following reaction on ice:
    2. 4-6 Fragment Assembly NEBuilder Positive Control
    Recommended DNA Molar Ratio Vector:Insert = 1:1
    Total Amount of Fragments 0.2 - 0.5 pmols
    X μl    		 10 μl
    NEBuilder HiFi DNA Assembly Master Mix 10 μl 10 μl
    Deionized H2O 10-X μl 0
    Total volume 20 μl 20 μl
  2. Incubate samples in thermocycler at 50°C for 60 minutes. Following incubation, store samples on ice or at -20°C for subsequent transformation.
  3. Transform NEB 5-alpha or 10-beta competent E. coli cells with 2 µl of chilled asssembled product, following the electoporation protocol.
Miniprep plasmid extraction protocol:
adapted from 'NEB - NEBuilder HiFi DNA Assembly reaction'
​
  1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1, before use vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
  2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of plasmid. If continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions, or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
  3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become cloudy. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue is gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
  4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.
  5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
  6. Centrifuge for 30–60 s. Discard the flow through.
  7. Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60s. Discard the flow through.
  8. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60s
  9. Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
  10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and centrifuge for 1 min.
Typical restriction digest:
example restriction reaction for SwaI
Component Volume
Template DNA 1 µg
10X NEBuffer r3.1 5 µl
Restriction enzyme 1 μl (10 units)
Nuclease-free Water to 50 μl
  1. Reactions were incubated for 30 mins at the enzyme-specific temperature.
  2. The reaction was terminated by heat-inactivation at 65◦C for 20 minutes.
PCR protocol:
BioMix™ Red
PCR was used to confirm HIFI cloning. The enzymes and reagents used were from Meridian Bioscience namely BioMix red and BioMix (no dye) was utilized unless otherwise indicated. All PCR reactions were performed in T100 Termal cycler (Biorad, USA).
A typical PCR setup is shown below:
PCR reaction
# BioMix red 5 μL Thermal cycling
1 10 μM Forward Primer 1.3 μL 95◦C 30 sec
2 10 μM Reverse Primer 1.3 μL 95◦C 10 sec
3 Miniprep (HiFi ~23 ng/μL and pAM4951 1 μL 60◦C 20 sec
4 Q5 High-Fidelity DNA Polymerase 0.25 μL 72◦C 30 sec/1Kb
5 - - Repeat thermal cycling of steps 2 through 4 30 times
6 ddH2O 1.4 μL 72◦C 2 min
Recipes
BG-11 media:
from 'UTEX BG-11 media'
Components
# Component Amount Stock Solution Concentration Final Concentration
1 NaNO3 10 mL/L 30 g/200mL dH2O 17.6 mM
2 K2HPO4 10 mL/L 0.8 g/200mL dH2O 0.23 mM
3 MgSO4•7H2O 10 mL/L 1.5 g/200mL dH2O 0.3 mM
4 CaCl2•2H2O 10 mL/L 0.72 g/200mL dh2O 0.24 mM
5 Citric Acid•H2O 10 mL/L 0.12 g/200mL dh2O 0.031 mM
6 Ferric Ammonium Citrate 10 mL/L 0.12 g/200mL dh2O 0.021 mM
7 Na2EDTA•2H2O 10 mL/L 0.02 g/200mL dH2O 0.0027 mM
9 Na2CO3 10 mL/L 0.4 g/200mL dH2O 0.19 mM
9 BG-11 Trace Metals Solution 1 mL/L - -
10 Sodium Thiosulfate Pentahydrate (agar media only,sterile) 1 mL/L 49.8 g/200 mL dH2O 1 mM
BG-11 Trace Metals Solution Components
# Component Amount Final Concentration
1 H3BO3 2.86 g/L 46 mM
2 MnCl2•4H2O 1.81 g/L 9 mM
3 ZnSO4•7H2O 0.22 g/L 0.77 mM
4 Na2MoO4•2H2O 0.39 g/L 1.6 mM
5 CuSO4•5H2O 0.079 g/L 0.3 mM
6 Co(NO3)2•6H2O 49.4 mg/L 0.17 mM
Method:
  1. To approximately 900 mL of dH2O add the first 9 components in the order specified while stirring continuously.
  2. Bring total volume to 1 L with dH2O.
  3. Cover and autoclave medium.
  4. Allow to cool then store at refrigerator temperature.