Team:Kent/Experiments


iGEM Kent 2015

Experiments & Protocols

Contents

Competent Cells
Transformation Protocol
Miniprep

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.