User:Tara K. Luckau/Notebook/Team ConGen/2011/10/14

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Revision as of 14:53, 17 October 2011 by Tara K. Luckau (talk | contribs) (Multiplexing and AutoDimer)
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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
20111014 Frag1.png

  • Here, average of intensity for column (rounded to nearest hundred), and ratio
20111014 Frag2.png

Best conditions

  • Scun7_25_SYRa
Scun7 - 0.25 µL
Scun10 - 0.15 µL
Scun16 - 0.2 µL
Scun22 - 0.2 µL
20111014 Frag3.png

  • Scun16_15_SYRa
Scun7 - 0.2 µL
Scun10 - 0.15 µL
Scun16 - 0.15 µL
Scun22 - 0.2 µL
20111014 Frag4.png

  • Scun19_30_SYRb (but Scun19 usually did not amplify to detectable levels)
Scun9 - 0.3µL
Scun15 - 0.25 µL
Scun19 - 0.3 µL
20111014 Frag5.png

Multiplexing and AutoDimer

  • Looked into whether there are tools available to check interactions between primers when multiplexing
  • Found AutoDimer by NIST (Butler 2004)
  • Ran SCOC primers, within each color lane, just out of curiosity
  • Only got one interaction at default settings (in the blue lane):
20111014 Primer.png
  • When threshold was decreased to 4, more interactions:
20111014 Primer2.png
  • 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