Team:MIT/miRNA Group Notebook Neon

Using the Neon to Transfect Cells · Benchling

Using the Neon to Transfect Cells

Introduction

Full manual here: https://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf These are notes from Wangui

Materials

  • DNA (midipreps ONLY, eluted in nuclease-free water!)
    • You need significantly more DNA than for a lipid-based transfection, ~1 ug per plasmid or so, but the concentration doesn't really matter.
  • PBS
    • There's a bottle in the iGEM TC drawer
  • 3 ml trypsin+EDTA, warmed to 37° in the dry bath
    • 7 ml complete media + (0.5 ml x # of transfections), warmed to 37° in the dry bath
      • 1 Neon "tube"
        • Cylindrical cartridge with a gold "bulge" on the side, in neon supplies box in iGEM drawer
      • Buffer E
        • In neon supplies box in iGEM drawer
      • Buffer R
        • In neon supplies box in iGEM drawer
      • Neon pipette tips
        • In iGEM drawer
      • The Neon
        • Sits on red cart in tissue culture. Comes with main computer, as well as a pipette station and the electroporation pipette.
      • 24 well plate(s)
        • Cells (growing in 10 cm plate, 70-90% confluent)
          • 1.7 ml centrifuge tubes (1)
            • Thin-wall 200 µl PCR strip tubes (1 per sample)

            Procedure

            • Preparing DNA
            1. Wipe down the Echotherm with 70% EtOH, place it in the hood, power it on and set it for 42°C.
            1. Prepare the DNA to electroporate: for each sample, mix up to 5 µg of DNA in a PCR tube. This is easiest if the tubes are in a rack; I like to use the 200 µl tip racks.
            • More DNA means more transfected cells! Unless you have a good reason, shoot to put at least 1 µg of each plasmid in each transfection.
            1. Place the PCR tubes in the Echotherm with the lids off to evaporate the water off of the DNA.
            • This can take ~30 minutes for 10 ul, which is why you should do this step first!
            • If you are pressed for time or your volumes are quite large, set the Echotherm to 50°. Don't go any higher.
            • Prepping Cells
            1. Retrieve plate from 37C. View under microscope. 70-90% confluence is a good range to transfect in.
            • For tHESC, one 10 cm plate should be enough cells for ~8-12 samples.
            • For HEK, one 10 cm plate would be enough for 36-48 samples.
            1. Aspirate the media off of the cells.
            1. Add 10 ml of PBS to wash.
            • Dribble down the sides to avoid disturbing cells
            1. Aspirate PBS.
            1. Add 3 mL of trypsin.
            • For tHESC, put in 37C, check on them occasaionally. Should take 7-10 mins.
            • For HEK, 3 mins at room temp is usually enough time.
            • Check under microscope to confirm cell movement.
            1. Add in 7 ml of complete media to quench the trypsin. Transfer to 15 ml conical.
            1. Centrifuge fo 5 mins at 150 xg.
            • During this spin, set up the transfection plate (below).
            1. Aspirate supernatant and resuspend in 10 ml of PBS.
            1. Take 15 ul to count in hemocytometer. count x 10^4 = # of cells/ml. # cells/ml * 10 ml of PBS = Total # cells.
            • For tHESC, this number may be ~200 uL
            1. Compute how much R buffer you'll need to add to reach a concentration of 10x7 cells/ml.
            • For a confluent plate of tHESC cells, this is ~200 ul.
            • Ex. Using the hemocytometer, I counted, on average, 10 cells in one four by four square. This means I have 1x10^5 cells/ml. In the PBS suspension, I have 1x10^6 cells total. I must resuspend later in 100 ul of R buffer to get a final concentration of 1x10^7 cells/ml.
            1. Compute how much R buffer you'll need and how many cells you'll need for the number of transfections you'll need to do.
            • You will need 12 ul cells + R buffer for each transfection.
            • You will need 100,000 (10^5) cells per transfection.
            1. If you have more cells than you'll need for the number of transfections, transfer a volume only the cells you need from the 15 ml conical to another 15 ml conical (or even a 2-ml centrifuge tube.)
            1. Spin down the cells again for 5 mins at 150 rcf.
            • During this spin, set up the electroporator (below)
            1. Aspirate PBS.
            • Try to get as much of the PBS off as you can. Be careful not to suck up your pellet.
            1. Resuspend in amount of R buffer you calculated in step 13. Transfer to a 2-ml eppendorf microcentrifuge tube.
            • Prepping 24 well
            1. Add 500 ul of complete media to each well in 24 well plate that you need.
            1. Place plate in 37°C for time being.
            • Setting up electroporator
            1. Plug in the Neon, leaving the "brain" on the red cart (there are outlets in the hoods as well as to the side, either is fine). Wipe down the pipette station and move it into the hood.
            1. Take a "tube" out of its bag. Place it in a tube rack and pipette 3 ml of buffer E into it.
            • Watch out! There is a slot down the side of the tube; make sure you pipette the buffer E down to the bottom.
            1. Set up the Neon pulse parameters.
            • Voltage: 1400
            • Width: 30 ms
            • Pulses: 1
            • (press the "database" button to find the tHESC preset, I think it's slot #9 on the second page).
            1. Transfer ~1.5 ml of PBS into a microcentrifuge tube and set aside.
            1. Put tip on electroporation pipette. Be very careful as the pipette delicate, expensive and easy to break.
            • DO NOT DO THIS UNLESS SOMEONE HAS SHOWN YOU HOW.
            • - First, push down to second stop on electroporation pipette. See how the little grippers extend out of the bottom of the pipette?
            • - Gently press pipette onto the tip. At this point, you are just trying to make the little latches on the pipette attach to the tip's gold plunger correctly.
            • - Slowly release the pipette plunger. Watch to see that the grippers grab the gold plunger and pull it up into the body of the pipette.
            • - Secure the tip into the pipette by pushing down firmly. You don't need to pipette up or down for this, just press the entire pipette down into the tip.
            • - Pick up the pipette + tip and make sure that when you pipette up and down, the clear part stays secure and the gold plunger moves up and down.
            • Zapping cells
            1. Bring the 24 well plate into the hood.
            1. Attach a clean aspirator tip to the vacuum line or saturate the one that's there with 70% EtOH; this will be used to clean the Neon tip between electroporations.
            1. Once DNA and electroporator is all set up, do the following one tube at a time.
            1. Add 12 ul of cells from resuspension (may need to pipette up and down again) to pcr tube with dna. Wait 30 seconds, then flick the side of the tube gently to mix.
            • You may need to pipette the cells suspended in R buffer up and down a time or two to resuspend them if they've fallen to the bottom of the tube.
            1. Take the electroporator pipette and pick up 10 ul of the cells+DNA mixture (10 ul is automatic, can't change it).
            1. Be careful to have ABSOLUTELY NO BUBBLES. Even the smallest bubble will cause a spark.
            1. Insert the electroporator pipette in the black station in the hood. Should click in place.
            1. Hit the start button. Watch the tip carefully; if it sparks, the transfection has failed and your cells have died.
            1. Once done, immediately pipette the electroported cells into their well. Jiggle or rock the plate to evenly disperse the cells in the wells.
            1. Rinse electroporator tip by pipetting some PBS up into the tip 3 times. Then put the orifice of the Neon tip against the opening of a a clean aspirator tip (vacuum ON) and pipette up and down a few times. Check to make sure there is no liquid left in the tip.
            1. NOTE: Because tips are difficult to put on the electroporator pipette, you will use the same tip for the remainder of the experiment. Be sure to rinse after every well though!
            1. Repeat from step 28 onwards for each sample.
            1. Store at 37C when done.
            • Cleaning up
            1. Everything can be disposed of normally
            • Pipette tip can be dislodged by pressing all the way down to the 2nd stop. The can go in regular hazardous wastes containers.
            • The cartridge for the little black station should have the E buffer that was in it poured out into the bleach bucket, then it could go in regular hazardous waste containers.