Team:IOANNINA/Protocols

Protocols

Lysogenic broths Plasmid Purification DH5a competent cells (NEB) transformation Agarose Gel Electrophoresis NEB HiFi DNA Assembly Colony PCR References

Note

The basic protocol that we followed for the clonetegration procedure was that established by Cui and Shearwin, 2017.

Lysogenic broths (LBs) for E.Coli DH5a cells

  • For 500 ml LB add in 500 ml dH2O 5 g tryptone , 2.5 g yeast extract , 5 g NaCl.
  • To make agarose plates add in 500 ml LB , 7.5 g agar.
    • For the the bacterial cultures the following antibiotics were used and added to the LB.
  • Chloramphenicol : final c= 25ug/ml
  • Ampicillin : final c= 100ug/ml
  • Kanamycin : final c= 50ug/ml or 20ug/ml

Protocol based on Monarch plasmid miniprep kit (NEB)

Materials

  • Monarch Plasmid Miniprep kit
  • Bacterial culture (1.5ml)

Protocol

  1. Centrifuge at 13.000 rpm (full) for 30 seconds, 1-5 ml (1.5 ml used) of the bacterial cultures keep pellet and discard supernatant.
  2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) . Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
  3. Add 200 μl Plasmid Lysis Buffer (B2) for cell Lysis. Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex. Incubate for one minute precisely.
  4. Add 400 μl of Plasmid Neutralization Buffer (B3), for lysate neutralization. Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex. Incubate again for 2 minutes.
  5. Spin (at high speed - 16,000 x g) for 5 minutes (avoid less than 2 minutes and over 5 minutes), for lysate clarification.
  6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
  7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1, which is used for RNA, protein and endotoxin removal (add a 5 minute incubation step before centrifugation if the DNA will be used in transfection). Wait for 1 minute and centrifuge for 1 minute. Discarding the flow-through is optional.
  8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
  9. Transfer column to a clean 1.5 ml microfuge tube. Transfer without touching flow-through !
  10. Add 30 μl DNA Elution Buffer (or Nuclease-free water / pH : 7–8.5) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

DH5a competent cells (NEB) transformation

We used the High Efficiency Transformation Protocol by NEB

Materials

  • NEB 5-alpha Competent E. Coli (High Efficiency)
  • Room temperature SOC
  • Vector
  • Heat Block (42°C)
  • Incubator (37°C)
  • LB-Agar plates with selection marker

Protocol

  1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
  2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipette 950 µl of room temperature SOC into the mixture.
  7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate (or centrifuge at 6000 rpm, for 10 minutes instead).
  8. Warm selection plates to 37°C.
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  10. Spread 50-100 µl (250 μl used) of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

Agarose Gel Electrophoresis

Protocol for 1% agarose gel electrophoresis

  1. Weigh 1 g of agarose and and in 100 ml 1x TAE buffer in a flask
  2. Heat up until the solution is homogeneous, avoiding boiling.
  3. Lower the temperature of the solution by adding water in the outer surface of the flask until the solution temperature is hot but manageable. Avoid mixing water with the solution.
  4. Prepare the electrophoresis chamber in which the gel will polymerize. Make sure that it is tightly sealed and that the gel supproting walls and the “comb” are well placed.
  5. Add 5 µL of Ethidium Bromide to the flask's solution and mix well.
  6. Pour the solution into the bed and clear all its bubbles with a tip. Let the solution to gelify (around 20-30 minutes).
  7. When the gel is ready, carefully pull out the “comb” and the gel supproting walls.
  8. Add enough 1x TAE Buffer so that it covers the gel.
  9. Prepare the loading samples with loading dye. Load the samples into the wells, as well as marker into the first well.
  10. Run the gel at 80 V for about 30 minutes.
  11. When the loading dye of the samples have reached approximately the 2/3 of the gel, the electrophoresis is complete. Put the gel into a UV chamber and take a picture of the results.

NEB HiFi DNA Assembly

Materials

  • NEBuilder HiFi DNA Assembly Master Mix
  • Ice
  • Insert
  • Leniarised Vector

Optimal Quantities Calculation

NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols of DNA fragments when 4–6 fragments are being assembled. 

To calculate the pmols of DNA use : 

  • pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
  • 50 ng of 5000 bp dsDNA is about 0.015 pmols
  • 50 ng of 500 bp dsDNA is about 0.15 pmols

Protocol

All the handling happened on ice.

For the assembly of one fragment to one vector we used 10 ul of NEBuilder HiFi DNA Assembly Master Mix with 0.04 pmoles of fragment and 0.02 pmoles of vector for a final ration vector:insert = 1:2.

  • For the control assembly we used 10 ul positive control and 10 ul NEBuilder HiFi DNA Assembly Mix.
  • Incubate samples in a thermocycler at 50 °C for 15 minutes
  • Following incubation store samples on ice or at -20 °C for subsequent transformation.
  • Transform NEB 5-alpha or 10-beta Competent E. coli cells with 2 μl of the chilled assembled product, following the High Efficiency Transformation Protocol as indicated previously.

Colony PCR

Materials

  • Agar plates with the appropriate antibiotic
  • ddH2O
  • Buffer 10X
  • DNTPs
  • MgCl2
  • Primers
  • Taq polymerase

Protocol

  1. Set up a number of PCR tubes with 50 µl H2O.
  2. Touch a yellow tip onto a colony, dip it into the first PCR tube, then streak it onto a new plate with a drawn template with numbered squares.
  3. Repeat this step for all colonies you need.
  4. Include two extra tubes: a negative and a positive control. The negative control contains only H2O, while the positive includes a colony, which is going to give a PCR product.
  5. Incubate the tubes for 10 min in 94oC - then place them on ice.
  6. Incubate the replicate agar plate at 30 or 37°C to grow again the colonies for future use.
  7. Set up a Master Mix as shown in the following table:
    8.  COMPONENT   CONCENTRATION 
     Buffer   10X 
     dNTPs   25mM 
     Primer forward   20pmoles 
     Primer reverse   20pmoles 
     MgCl2   25mM 
     Dilluted culture   2ul 
     Taq polymerase   0,2ul 
     H2O   Final volume=50ul 
  8. Add 20ul of the Master Mix to each tube.
  9. Set the following PCR program:
    10.  Step   Time   Temperature (oC) 
     Initial Denaturation    2 min   94
     30 cycles  45 sec   94
     30 cycles  30 sec   55
     30 cycles  40 sec   72
     Final extension   5 min   72
     Hold   -  4
  10. Run the program.