Andy Chen:Micromanipulation
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Micro-manipulation Technique Notes[edit]
General Protocol[edit]
- Prepare pipette tips, tubes for collected samples, and a tube of medium (PBS, H2O, etc). UV everything 10-15 mins.
- Transfer desired amount of medium to each sample tube (for example, fill tube with 1 μL if doing a subsequent 20 μL MDA amplification)
- Load a tip into the pipetter and pre-load the tip with medium. To do this, move the pressure to slightly positive (+10 to +20), immerse the tip in the tube of medium, move the pressure to slightly negative (-5 to -10), wait for the pressure to equalize (it will stop clicking), then move the pressure back to zero.
- Move the tip to the desired well. Take a picture of the fluorescence for reference.
- Apply a positive pressure to eject some of the fluid into the well (enough to cover the bottom of the well). Move the tip around to help loosen the DNA-bead complex. Apply a negative pressure to extract the DNA. Check fluorescence to see if DNA was extracted adequately. If not, repeat ejection-extraction a few more times to collect as much DNA as possible.
- Move tip to home and eject the collected DNA into sample tube by first applying a large positive pressure (+50, for example) and then hold the blue [INJECT] button for a few seconds to insure all the DNA was ejected.
- Tips around 20-50 μm in diameter are ideal.
- When filling a dry microwell with medium, there seems to be a threshold to getting the medium out of the tip. Thus positive pressure needs to be applied carefully to prevent flooding and possible contamination. Pressure should be increased slowly. If there appears to be a bulge of medium on the tip, try to move the tip around on the bottom of the well to get some liquid into the well, making it easier to add medium.
- I found it easier to move to the general location of the well using the [0] setting, then changing it to [1] for finer movements. [0] seems to be affected by acceleration, so if you aren't careful, it is easy to over move it and smash the tip into the well.
General Protocol[edit]
- Prepare tips, sample tubes, medium tubes, tubes for diluting cells (if necessary), slide and coverslip. UV everything 10-15 minutes.