Invitrogen and Holmes, Dawn 1-2
Cloning with the Invitrogen TOPO Cloning Kit
Ligation
1. Set a heat block to 42oC for later use.
2. Remove the provided SOC media and the premade LB Ampicillin X-Gal plates from the
4oC. Leave standing at room temperature until needed, so that they can warm up to room
temperature before use.
3. In a sterile 0.5mL tube mix:
0.5uL salt solution
0.5uL TOPO vector
2uL gel extracted PCR Product
4. Allow this mixture to incubate at room temperature for between 30 minutes and 2 hours,
so that the insert can ligate into the vector. The larger the insert is the longer this
incubation should be carried out. For a very simple and small insert, such as the 500 base
pair 16S PCR product this incubation can be reduced to 15 minutes.
5. Continue with the Chemical Transformation or Electroporation steps below.
Chemical Transformation
1. Remove the Top10 chemically competent cells from the –80oC freezer and thaw on ice.
You will need 1 tube of cells per 2 cloning reactions.
2. Split the tube of cells into 2 tubes with 30uL each, either 1.5mL or 2mL tubes can be
used.
3. Add 2uL of the TOPO cloning reaction prepared in step 3 to one tube of 30uL cells.
Gently mix the cells by flicking the tube with your finger.
4. Incubate the cells on ice for 20-30 minutes.
5. Using the heat block set up earlier incubate the cells at 42oC for 30 seconds then place at
room temperature. This will heat shock the cells and cause them to take up the vector.
6. Add 125uL of the room temperature SOC media to each tube of cells.
7. Continue with the Recovery steps below
Invitrogen and Holmes, Dawn 2-2
Electroporation
1. Remove the Top10 electrocompetent cells from the –80oC freezer and thaw on ice. You
will need 1 tube of cells per cloning reaction.
2. Add 2uL of the TOPO cloning reaction prepared in step 3 to one tube of cells. Gently
mix the cells by flicking the tube with your finger.
3. Incubate the cells on ice for 20-30 minutes.
4. Carefully pipette up the cells and transfer to a cold electroporation cuvette. Be sure to
put the cells into the very bottom of the well where the well narrows and becomes very
thin.
5. Place the cuvette into the electroporation handle (there is only one orientation that the
cuvette with fit this handle).
6. Carefully slide the handle into the electroporation unit until the cuvette snaps into place
between the two metal pieces.
7. Check to see that the electroporation unit is set to Ec1 and press the pulse button. You
will hear a short buzz for the electrically pulse, this will only take a few seconds
8. Add 1mL of the room temperature SOC media to each cuvette of cells, then transfer to a
1.5mL or 2mL sterile tube.
9. Continue with the Recovery steps below
Recovery
1. Recover the cells shaking at 37oC for 30 minutes to 1 hour. Be sure to tape the tubes on
their side to the rotator, thereby ensuring aeration.
2. In a sterile work space with a Bunsen burner pipette 100uL of the recovered cells onto the
room temperature LB Ampicillin X-Gal plates.
3. Incubate the plates upside down at 37oC overnight (16-20 hours)
4. Move plates to 4oC for storage. If the plates are to be stored for more then a few weeks
cut a pick of parafilm and stretch around the edges of the plate to seal the plate from
drying out or growing mold. The cold temperature will also help darken the blue
colonies for easy picking.
5. When you wish to use the colonies, you will need to pick only the white colonies as these
colonies have the lacZ gene disrupted by the insert.