Team:Kent/Experiments


iGEM Kent 2015


Protocols

Contents

Competent Cells
Transformation Protocol
Miniprep
PCR
Ligation
AFM Imaging
Gibson Assembly

Competent Cells

Overview

Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which foreign DNA can be passed through easily. E. coli cells that have been specially treated to transform efficiently.

Materials

  • 3ml 1M MnCl2
  • 15ml 1M CaCl2
  • 60ml 50mM MES
  • 45ml glycerol
  • 177ml ddH2O

    • Procedure

    1. Overnight culture of VS45 cells are back-diluted to OD600 0.1 in 250 ml LB broth
    2. The cells are then grown at 37˚C to OD600 0.6 and then harvested by centrifugation.
    3. The cells are resuspended in 100 ml of prechilled buffer and incubated on ice for 60 minutes.
    4. Harvest again by centrifugation (at 4˚C), and resuspended in 5 ml of pre-chilled buffer.
    5. The resuspended cells can then be aliquoted (on ice), frozen using dry ice or liquid nitrogen, and stored at -80˚C.

    Transformation Protocol

    Overview

    Transformation is the process by which a foreign DNA is introduced into a cell.

    Materials

  • Resuspended DNA
  • Competent cells
  • 2ml tube
  • 42˚C water bath
  • Petri dishes with LB agar and appropriate antibiotic
  • 37˚C incubator
  • 10pg/ul RFP Control

    • Procedure

    1. Thaw the competent cells on ice
    2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
    3. Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently.(Make sure to keep the competent cells on ice. )
    4. Add 1 µL of the RFP Control to your control transformation.
    5. Close tubes and incubate the cells on ice for 30 minutes.
    6. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
    7. Incubate the cells on ice for 5 minutes.
    8. Add 200 μl of SOC media (making sure that the broth does not contain antibiotics and is not contaminated) to each transformation
    9. Incubate the cells at 37˚C for 2 hours while the tubes are rotating or shaking. 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
    10. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
    11. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
    12. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. (Incubating for too long starts to break down the antibiotics and un-transformed cells will begin to grow.)
    13. Pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
    14. Count the colonies on the 20 μl control plate.

    Miniprep

    Overview

    The Miniprep is for purification of molecular biology grade plasmid DNA, this provides a rapid method to purify plasmid DNA using silica membrane column.

    Materials

  • Buffer P1
  • Buffer P2
  • Buffer N3
  • Buffer PB
  • Buffer EB
  • Buffer PE

    • Procedure

  • Add the provided RNase A solution to Buffer P1.
  • Mix the solution and store at 2–8°C
  • Add ethanol (96–100%) to Buffer PE before use
    1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
    2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer it to a microcentrifuge tube.
    3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
    4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
    5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
    6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through.
    7. Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
    8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through
    9. Centrifuge for 1 min to remove residual wash buffer.
    10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
    11. Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

    PCR

    Overview

    PCR is a method to amplify sections of DNA fragments

    Materials

  • 50µl PCR reaction

    • 300nM of forward and reverse primers

    25µl 2x Master Mix

    21µl sterile MQ H2O

    Procedure

  • Pick a colony and resuspend in 50µl of MQ H2O
  • Take 1µl of the cell suspension and add to 50µl PCR reaction
  • PCR cycles:
  • 1. 95º for 5 minutes
  • 2. 95º 19 seconds
  • 3. 50º for 30 seconds
  • 4. 68º for 1.5 minutes
  • 5. 68º for 5 minutes
  • steps 2-4 last for 35 cycles

    • Ligation

    AFM

    Overview

    A method of viewing the topography of a sample using Atomic Force Microscopy, generating a 3-D image

    Materials

  • Chloramphenicol and Amp combined plates (non-inducing)
  • Chloramphenicol and Amp combined plated, with 20% w/v Arabinose and 1mM IPTG (inducing)
  • 1x PBS
  • 0.5ml water

    • AFM Preparation procedure

  • Grow VS45 containing PVS72 on an inducing plate and a non-inducing plate for 5 days at 22ºC
  • Pipet 25µl 1x PBS onto a spot of bacteria containing PVS72 on both an inducing plate and a non-inducing plate
  • Pipet the PBS up and down 5 times
  • Transfer 20µl of each resuspension into an eppendorf tube
  • Place 10µl of each sample onto a freshly cleaved mica
  • Incubate for 40 minutes
  • Wash with 0.5ml of water

    • Gibson Assembly

    Overview

    Gibson assembly is a method of joining DNA fragments in a single reaction.

    Materials

  • 2-10µl of PCR fragment + linearized vector (25-100ng of vector and at least 2-fold excess inserts)
  • 10µl of Gibson Assembly Master Mix
  • Deionised H2O

    • Assembly Procedure

  • Add 2-10µl of PCR Fragments + linearized vector to 10µl of Gibson Assembly Master Mix
  • Add Deionised H2O as necessary to bring the total reaction volume to 20µl
  • Incubate the samples at 50ºC in a thermocycler for 15 minutes
  • Store the samples at -20ºC
  • Transform the product into competent cells using the Gibson Assembly Transformation Protocol

    • Gibson Assembly Transformation Protocol

  • Thaw competent cells on ice
  • To the competent cells, add 2µl of the chilled assembly product
  • Mix the Assembly product with the competent cells by pipetting up and down 4-5 times
  • Incubate the mixture on ice for 30 minutes
  • Heat shock at 42ºC for 30 seconds
  • Return the tubes to ice for 2 minutes
  • Add 950µl of SOC media to the tube
  • Incubate at 37ºC for 60 minutes, whilst shaking
  • Warm appropriate selection plates to 37oC
  • Spread 100µl of cells onto the plates
  • Incubate overnight at 37ºC