SCOC MP1
Fragment Analysis
Summary of fragment analysis results
- Numbers indicate intensity of peak for each marker (or average of peaks, if heterozygous)
- At bottom, indicates ratio of each lane
- Blue - Scun7:Scun10:Scun16:Scun22 intensity
- Green - Scun9:Scun15:Scun19 intensity
- Here, average of intensity for column (rounded to nearest hundred), and ratio
Best conditions
- Scun7 - 0.25 µL
- Scun10 - 0.15 µL
- Scun16 - 0.2 µL
- Scun22 - 0.2 µL
- Scun7 - 0.2 µL
- Scun10 - 0.15 µL
- Scun16 - 0.15 µL
- Scun22 - 0.2 µL
- Scun19_30_SYRb (but Scun19 usually did not amplify to detectable levels)
- Scun9 - 0.3µL
- Scun15 - 0.25 µL
- Scun19 - 0.3 µL
Multiplexing and AutoDimer
- Looked into whether there are tools available to check interactions between primers when multiplexing
- Found AutoDimer by NIST (Butler 2004)
- http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do
- Ran SCOC primers, within each color lane, just out of curiosity
- Only got one interaction at default settings (in the blue lane):
- When threshold was decreased to 4, more interactions:
- I think it's useful to decrease the threshold to 4 or 5 to have program spit out more interactions, and be able to decide for myself which ones I care about
Next Steps
- optimize within yellow lane (Scun3, Scun11, sar80)
- see if green and yellow can multiplex (same PCR conditions)
- diversity panel on all lanes, and post-PCR mix
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