Mapping 250K/500K SNP assay

LIGATION PROCEDURE
PRE-PCR CLEAN AREA
Table C.4
254 GeneChip ® Mapping 500K Assay Manual
Program the thermal cycler in advance. Switch on the thermal
cycler 10 minutes before reactions are ready so that the lid is
heated.
Ligase buffer contains ATP and should be thawed/held at 4°C. Mix
ligase buffer thoroughly before use to ensure precipitate is resuspended.
Avoid multiple freeze-thaw cycles, according to
vendor’s instructions.
1. Depending on the restriction enzyme used, prepare the following
Ligation Master Mix ON ICE (for multiple samples make a 5%
excess):
Nsp I Sty I
Reagent 1 Sample Final Conc.
in Sample
Reagent 1 Sample Final Conc.
in Sample
Adaptor Nsp I (50 µM) 0.75 µL 1.5 µM Adaptor Sty I (50 µM) 0.75 µL 1.5 µM
T4 DNA Ligase buffer
(10X) *
T4 DNA Ligase
(400 U/µL)
2.5 µL 1X T4 DNA Ligase buffer
(10X) *
2 µL 32 U/µL T4 DNA Ligase
(400 U/µL)
Total 5.25 µL Total 5.25 µL
2.5 µL 1X
2 µL 32 U/µL
* Contains ATP and DTT. Keep on ice. * Contains ATP and DTT. Keep on ice.