Nucleofection

Nucleofection kdorfman Tue, 09/25/2012 - 18:08
Nucleofection handout.pdf
Amaxa Nucleofector II Manual

Mirus

Mirus kdorfman Fri, 12/06/2019 - 14:36

general summary

100 uL Mirus reagent
2 ug DNA(up to 20 uL DNA solution)
1 - 5 million cells/mL Lonza Knowledge Center

Protocol

  • Warm medium in flask and coverslip-bottom dish
  • Mix DNA and Mirus reagent; warm up
  • Trypsinize cells:
    • remove medium
    • rinse with warm PBS
    • add 0.5 mL trypsin
    • incubate till all cells are in suspension (at least 3 min)
    • add 1 mL COLD medium
    • mix well by pipetting up and down
  • transfer all to sterile 2 mL tube
  • spin 500 x g 3 minutes
  • resuspend cells in Mirus/DNA solution
  • put all into sterile cuvette, close
  • Put cuvette into Nucleofector
  • Choose program1
  • Press the button
  • Use special dropper to transfer one drop to the coverslip dish and the rest to the waiting flask.

Mirus nucleofection kit

  1. Cell type Program #
    B16 P-031
    3t3 U-030
    HCT116 D-032
    HeLa I-013
    LLCPk-1 X-001
    MEF T-020, A-023

    ↩︎

ml052_ingenio_electroporation_products.pdf
Nucleofection handout_0.pdf
nucleofector manual_0.pdf

Cell Density

Cell Density kdorfman Wed, 09/29/2021 - 16:47
See estimating cell numbers
If cells are % confluent
in a sq cm flask
there are roughly million cells in the flask

selection of nucleofected cells

selection of nucleofected cells kdorfman Thu, 10/03/2019 - 20:47

Addgene plasmid selection

Addgene plasmid selection kdorfman Sat, 05/30/2020 - 13:52

When cells are medium-light, replace medium with 0.5 g/L G418 (= 0.5 mg/mL)

Next day, replace with medium with 1 g/L (=1 mg/mL)

Next day, 2 g/L (=2 mg/mL)

Solution is 50 mg/mL

Dilute 10 uL per mL to get 0.5 mg/mL

Dilute 20 uL per mL to get 1 mg/mL

Dilute 40 uL/mL to get 2 mg/mL

Making a Stable Cell Line Expressing Your Favorite Gene

Making a Stable Cell Line Expressing Your Favorite Gene margaret Fri, 10/21/2011 - 14:38
  1. Make a death curve. This is to determine the minimal concentration of antibiotic that kills cells that do not have the resistance gene.
  2. transfect the cells with the plasmid containing the gene that you want to express in the cells (see Amaxa protocol).
  3. incubate the cells for about 48 hours after transfection so that the gene is expressed, and the resistence gene is expressed.
  4. add the appropriate dose of antibiotic (this is the selection step). The cells that do not contain the resistance gene (i.e. that were not successfully transfected) will die. Change the medium every 2 or 3 days to remove dead cells. Replace medium with fresh medium containing the antibiotic.
  5. split the cells as needed. Use medium with antibiotic. Make some coverslips to see if the cells are expressing the gene of interest.

After ~2 weeks any cell without the transgene and selectable marker should have died. The remaining cells are a mixed population – different cells might express different levels of the transgene. These can be used for preliminary experiments.

Making a clonal cell line

  • A Clonal cell line contains cells that are derived from a single cell, and thus are a clone. They will all be genetically identical; this can be very helpful for experiments.
  • a. Trypsinize your 'mixed population' of cells; keep some growing in a flask and freeze some in case your cloning does not work the first time and you need to repeat the procedure.
  • b. take 50ul of the trypsinized cells, dilute again into 1 ml medium. Add 10 -150 ul of this dilute solution of cells to 100mm dishes containing growth medium. Look under the tissue culture scope. The cells should be very dilute – one or just a few cells per field of view. If they are too dense or dilute, adjust accordingly. You want each individual cell to land on the plastic dish and grow into a colony of cells that is separate from other colonies on the dish.
  • c. wait about 1 week for the colonies to grow.
  • d.isolate individual colonies. These are potential clonal cell lines.
    • 1. prepare cloning rings by smearing a small amount of grease on one end; make about 10. Have sterile forceps ready; have trypsin ready. Have a 24 well plate ready with some medium in the number of wells that you are planning to use.
    • 2. remove medium from dish.
    • 3. rinse with PBS--; leave a little bit so the cells don't die.
    • 4. tilt the dish to see the colonies; place a cloning ring over nice large and well separated colonies. Press down firmly. Don't let the ring slide around. Add ~100ul of trypsin to each ring; wait a few mins. Pipette cells up and down (in the ring) add the released cells to one well of a 24 well plate. Each well now has a colony of cells – a potential cell line. When they grow, split each well – place some cells in a well of a six well plate to grow some more and put some in a Matek dish – be sure to label carefully so you know which is which. When the cells in the Matek dish are grown – check them in the fluorescence microscope. If they all have the same level of fluorescence and are healthy looking, they are a potential line (which you can further characterize with a Western Blot to quantify the level of expression).

    Toss any cell lines that are not healthy or have too much or too little fluorescence.

    Lonza Amaxa

    Lonza Amaxa kdorfman Fri, 12/06/2019 - 14:34

    Nucleofection with Lonza Amaxa

    Order VCA-1005 Cell Line Nucleofector Kit L (25 reactions $347)

    Transfection Protocol

    • Warm medium in MatTek dishes to equilibrate CO2
    • Warm additional 0.5 mL medium in sterile microfuge tube
    • Make mucleofection solution, RT, per flask:
      • 19 µL supplement 1
      • 81 µL reagent L for LLCPk (Kit R for 3t3, HeLa)
    • Remove medium from flask
    • Rinse with PBS
    • Add trypsin
    • Incubate ~5 min, or until cells are floating
    • Add cold medium
    • Transfer to sterile 15 mL centrifuge tube
    • Spin 10 min, slow speed
    • Remove medium. Pellet should be soft
    • Loosen pellet by flicking tube
    • Add all 100 µL nucleofection solution plus the DNA (one of the following):
      • 2 µg plasmid DNA
      • 5 µg BAC
      • 5 µL siRNA (20 µM)
    • Pipet up and down to mix
    • Transfer to nucleofection cuvette
    • Insert into nucleofector device, choose program
      • X001 for LLCPK
      • I-013 for HeLa
      • U-030 for 3t3
    • Resuspend cells in 500 µL warm medium
    • Distribute in drops on coverslips of MatTek dishes
    nucleofector manual.pdf