Make sure that the contents of each tube are clear of any precipitate and are thoroughly mixed before setting up the reaction:

• Mix the contents of the following tubes by vortexing: L Fragment Buffer (LFB), Elution Buffer (EB), Ligation Adapter (LA), and the NEBNext Quick T4 DNA Ligase
• Mix the tube of Ligation Buffer (LNB) by pipetting up and down
• Look at the tube contents to make sure there is no precipitate formed
• Spin down the tubes briefly in a microfuge
• Put the tubes on ice immediately after thawing and mixing

Take Tube 3 with 60 µl of the Lambda DNA library and add 25 µl Ligation Buffer (LNB).

Add 10 µl of the NEBNext T4 DNA Ligase to the tube.

Add 5 µl of Ligation Adapter (LA) to the tube.

Mix the contents of the tube by flicking the tube with your finger. Spin down briefly in a microfuge.

Incubate the reaction for 10 minutes at room temperature.

Add 40 µl of resuspended AXP to the tube with the Lambda DNA library (Tube 3), and mix by pipetting up and down.

Put the tube in a Hula mixer, and leave to incubate for 5 minutes.

Take Tube 3 with the Lambda DNA sample off the Hula mixer, and spin down in a microfuge.

Place the tube in a magnetic rack, and wait for the beads to collect in a pellet near the magnet, and the solution to become clear.

Keep the tube on the magnet to pipette off and discard the supernatant. Take care to not disturb the pellet.

Add 250 µl of L Fragment Buffer (LFB) to Tube 3. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Pipette off and discard the L Fragment Buffer (LFB).

Repeat the previous step:
Add 250 µl of L Fragment Buffer (LFB) to Tube 3. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Pipette off and discard the L Fragment Buffer (LFB).

Take the tube off the magnetic rack and spin down briefly in a microfuge.

Place the tube back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet. There may be a small amount of residual liquid at the bottom of the tube. Pipette off and discard this liquid.

Remove the tube from the magnetic rack, add 15 µl Elution Buffer (EB) to the tube, and resuspend the beads by flicking the tube.

Incubate the reaction for 10 minutes at room temperature.

Place the tube back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet.

Pipette off the 15 µl of eluate from Tube 3 and transfer it to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 4). Discard Tube 3 with the pellet.