-
Set up the following biotin tagging reaction in a 0.2 ml thin-walled PCR tube:
Reagent |
Volume |
cDNA template |
10 ng, x μl |
[Btn]Fwd_3580_partial_read1_defined, 10 μM |
2 μl |
Rev_PR2_partial_TSO_defined, 10 μM |
2 μl |
Nuclease-free water |
21-x μl |
LongAmp Hot Start Taq 2X Master Mix |
25 μl |
Total |
50 μl |
-
Amplify using the following cycling conditions:
Cycle step |
Temperature |
Ramp rate |
Time |
No. of cycles |
Initial denaturation |
94°C |
max |
3 min |
1 |
Denaturation
Annealing ramp-down
Annealing
Extension |
94°C
66°C down to 58°C
58°C
65°C |
max
0.2°C/s
max
max |
30 sec
40 sec
50 sec
6 mins |
4 |
Final extension |
65°C |
max |
10 min |
1 |
Hold |
4°C |
- |
∞ |
- |
-
Resuspend the AMPure XP beads by vortexing.
-
Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
-
Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
-
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
-
Repeat the previous step.
-
Briefly spin down and place the tubes back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellet to the point of cracking.
-
Remove the tube from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 minutes at room temperature.
-
Pellet the beads on a magnet until the eluate is clear and colourless.
-
Remove and retain 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.