Mapping 250K/500K SNP assay

214 GeneChip ® Mapping 500K Assay Manual
Troubleshooting Guide for the GeneChip ® Mapping 500K
Assay
Problem Likely Cause Solution
Faint/absent bands on PCR gel
Both samples & positive
control affected.
Problem with master mixes or
individual reagents.
Ensure all reagents added to master mixes
and enzymes are stored at –20°C. Work
quickly with enzymes and return to –20°C
directly after use to prevent loss of activity.
Failed restriction digest. Use restriction enzyme to digest a known
good DNA sample. Run gel to confirm
restriction enzyme activity.
Failed adaptor ligation reaction. Confirm enzyme activity.
Reduced adaptor ligation efficiency
due to adaptor self-ligation, DNA religation.
Ligase buffer contains ATP and should be
defrosted/ held at 4°C. Mix ligase buffer
thoroughly before use to ensure
precipitate is re-suspended. Avoid
multiple freeze-thaw cycles. Try a fresh
tube of buffer.
To prevent self-ligation of adaptor work
rapidly and add DNA ligase last.
Failed PCR reaction. Check PCR reagents. Take care with
preparation of master mixes and ensure
accurate pipetting and thorough mixing.
Reduced PCR reaction yield – non
optimal PCR conditions.
Ligation mix not diluted prior to PCR
reaction.
Incorrect concentration of
nucleotides.
Used Nsp adaptor for Sty digest, or
vice versa.
Use a validated thermal cycler, check PCR
programs. Use recommended thin walled
reaction tubes.
Thoroughly mix PCR reaction.
Ligation mixture diluted 1:4 with molecular
biology grade water to remove potential
inhibitors and maintain optimal pH and
salt concentration.
Check dNTP stock concentration and
vendor.
Repeat Ligation step with correct adaptors.