Mapping 250K/500K SNP assay

chapter 4 | 96-Well Plate Protocol 57
Preheat the Thermal Cycler Lid
Power on the thermal cycler to preheat the lid. Leave the block at room
temperature.
The lid must be preheated before samples are loaded.
PREPARE THE LIGATION MASTER MIX
Keeping all reagents and tubes on ice, prepare the Ligation Master Mix
as follows:
1. To the 2.0 mL Eppendorf tube, add the following reagents based
on the volumes shown in Table 4.11 (for Nsp) or Table 4.12 (for
Sty):
• Adaptor (Nsp or Sty)
• T4 DNA Ligase Buffer (10X)
2. Remove the T4 DNA Ligase from the freezer and immediately
place in the cooler on ice.
3. Pulse spin the T4 DNA Ligase for 3 sec.
4. Immediately add the T4 DNA Ligase to the master mix; then place
back in the cooler.
5. Vortex the master mix at high speed 3 times, 1 sec each time.
6. Pulse spin for 3 sec.
7. Place the master mix on ice.
8. Proceed immediately to Add Ligation Master Mix to Reactions.
Table 4.11
Nsp I Ligation Master Mix
Reagent 1 Sample 96 Samples
(15% extra)
Adaptor Nsp I (50 µM) 0.75 µL 82.8 µL
T4 DNA Ligase Buffer (10X) 2.5 µL 276 µL
T4 DNA Ligase (400 U/µL) 2 µL 220.8 µL
Total 5.25 µL 579.6 µL