- Before you do lysis, make sure that you have put your plate into LIMS for the LIMS Submission.
- The plate that is used for lysis has the same number of wells and positions as the matrix box. Label the lysis plate with the box number, lysis, the date and your name.
- For one plate, dispense 5 ml of Vertebrate or Invertebrate Lysis Buffer (depending on if you are working with verts or inverts) into a white tray. In the Science Complex, there are different labelled pipettes in the drawer for vert and invert lysis buffer. Never pipette by mouth. In BIO, take off the black cap, lift up the white arms, then pull the white top up and as you press it down lysis buffer comes out. Add 500ul of Proteinase K (it will be in a white box in the freezer) to the lysis buffer in a white tray. Rock it back and forth to mix it.
- Using a multi-channel pipette transfer 50 μl of the mixture you just made into each well of the 96 well plate using 200ul tips. In BIO you have to use the electronic multichannel-pipette. Unplug it. Press ON. Press Volume, and set it to 700. Then press Dispense and set it to 50. Press enter until it beeps, and then press enter again. You do not have to change tips each row. Cover the plate with cap-strips (not very tight, just press hard on A and G). Plug the pipette in, and turn it off.
- Flame Sterilization is used for insect legs, and other dry tissue that is less likely to cause contamination. When subsampling fleshy tissue like fish or other non-insects, the tissue tends to stick to the tools so for those types of tissue, we use Eliminase for Sterilization. If the tissue does not need to be subsampled and lysis is going to be done in plate, then add the lysis solution to the plate directly after leaving the tissue plate in the incubator, uncovered but with a Kim Wipe over the wells until all the ethanol has evaporated.
- When you are done, make sure that you have put the Lysis plate onto LIMS.
Return to Barcoding in the Hanner Lab Wiki.
Next you have to extract the DNA with either the Robotic DNA Extraction in BIO.
Updated April 27 2009