Trypsin Preparation

From LPDwiki

About this protocol: Trypsin is a digestive protease that is used to cleave cell adhesion proteins. This process, known as trypsinisation, is used primarily for the passaging of cell cultures. Here the protocol for the preparation of the 1X trypsin solution used in the cell passaging process is presented. [1]

Necessary Materials

1. Trypsin (10x):

  • Trypsin is purchased from Invitrogen (15400-054) and should be separated to 5 mL aliquots upon arrival.
  • Trypsin should be stored at -80°C

2. Phosphate Buffered Saline (PBS) without Ca or Mg [-]

  • PBS[+] and PBS[-] are purchased from Invitrogen (GB122).
  • PBS should be stored at 4°C
  • Use only PBS solutions marked STERILE for this protocol

Procedure

Important: The Biosafety Cabinet (BSC) must be used for the preparation of trypsin. Please follow the Biosafety Cabinet Start-up procedure before beginning this protocol.

1. Thaw 10x trypsin in 4°C the night before starting this protocol.

Inside the BSC

2. Add 40 mL of PBS to a conical tube.

3. Add 5 mL of PBS to the 5mL aliquot of trypsin and agitate the solution (via pipetting) to ensure mixing.

4. Add the 10 mL typsin+PBS solution into the 40 mL of PBS prepared in Step 1.

5. Agitate the solution (via pipetting) and cap tightly.

6. Label with

  • Date made:
  • Solution Name: (1X Trypsin in PBS, Sterile)
  • Your name:

Outside the BSC

7. Place the newly made trypsin solution into 4°C storage.

Cleaning Up

1. Ensure the remaining PBS is tightly capped and place into 4°C for storage.

2. Dispose of used conical tubes and pipettes into the biological waste bin.

3. If you are done with work in the BSC, follow the Biosafety Cabinet Shutdown procedure.