Mapping 250K/500K SNP assay

PCR PROCEDURE
PRE-PCR CLEAN ROOM
appendix C | Low Throughput Protocol 259
The PCR reaction is sensitive to the concentration of primer used. It
is critical that the correct amount of primer is added to the PCR
reaction to achieve the correct distribution of fragments (200 to
1100 base pairs) in the products. Check the PCR reactions on a gel
to ensure that the distribution is correct (see Figure C.2).
1. Prepare the following PCR Master Mix ON ICE (3 PCR reactions
per sample) for Nsp I or Sty I ligation reactions and vortex at
medium speed for 2 seconds (for multiple samples make a 5%
excess):
Table C.7
Stock Reagent 1 PCR 3 PCR Final Conc. in
Sample
H 2O 39.5 µL 118.5 µL
Clontech TITANIUM Taq
PCR Buffer (10X)
10 µL 30 µL 1X
G-C Melt (5 M) 20 µL 60 µL 1 M
dNTP (2.5 mM each) 14 µL 42 µL 350 µM (each)
PCR Primer 002 (100 µM) 4.5 µL 13.5 µL 4.5 µM
Clontech TITANIUM Taq
DNA Polymerase (50X)
2 µL 6 µL 1X
Total 90 µL 270 µL
90 µg of PCR product is needed for fragmentation.