User:Tara K. Luckau/Notebook/Team ConGen/2013/02/27

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==CNTI Acos5==
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==SCOC MP2 Optimization==
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===PCR===
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* uniplex SW markers to see quality of peaks and range of alleles
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* [[Image:20120110_PCRa.png|800 px]]
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* multiplex SW markers as first glance of how multiplex behaves
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===Gel===
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===Results===
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* Frag data: PPTX
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* also using frag data from FHVZ (late November 2012)
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** (apparently, I already did this PCR of uniplexing each SW marker, then throwing it all in together ... oops)
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{| {{table}} style="text-align: center"
{| {{table}} style="text-align: center"
|-
|-
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! Pour !! Load !! Run
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! Marker !! SYR Genotype !! TP3 Genotype !! Uniplex comments !! Multiplex comments
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|-
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| 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V
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| style="background: yellow" | SW614-A1 (NED) || 410 415 || style="background: Silver" | [poop] || tall @ 8000, very clean || short @ 300, clean
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|-
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| style="background: royalblue" | SW614-A4 (FAM) || 395 396 || 396 398 || tall @ 15000 || short @ 300
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|-
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| style="background: red" | SW614-A6 (PET) || style="background: Silver" | [poop] || 255 275 || tall @ 8000 || good @ 1500
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|-
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| style="background: green" | SW614-A7 (HEX) || 252 252 || 248 248 || tall @ 14000 || style="background: Silver" | no amplification
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|-
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| style="background: green" | SW614-B1 (HEX) || 316 316 || 299 316 || tall @ 16000, one replicate of SYR poop || good @ 1200
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|-
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| style="background: royalblue" | SW614-B6 (FAM) || 308 310 || 308 310 || tall @ 11000 || style="background: Silver" | no amplification
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|-
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| style="background: royalblue" | SW614-B10 (FAM) || 188 194 || 186 198 || tall @ 12000, one replicate of SYR violates slope rule || style="background: Silver" | no amplification
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|-
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| style="background: yellow" | SW614-B12 (NED) || 285 285 || 255 274 || tall @ 14000 || good @ 2000
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|-
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| 27 µL GelRed || 6 µL ladder || 45 minutes
 
|}
|}
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[[Image:20120110_Gela.png|800 px]]
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* regarding sar80: after reviewing past PCRs using sar80, seems monomorphic in diversity panel, doesn't multiplex well ... no need to use this finicky marker
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* mispriming everywhere - primer pair not usable for CNHY
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* lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions
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* favorable conditions indicated by [[Image:greenhappyface.png|25 px]]
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* definitely move forward with SW614-A4, SW614-B1 and SW614-B12 as SCOC MP2
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* may consider adding SW614-A6, just to include that color. I can possibly imagine that SYR's genotype is 260 263 (with lots of stutter), which means other samples may be just fine. Might as well include the marker, and if scoring ends up being too difficult, then we'll dump the marker.

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Contents


SCOC MP2 Optimization

  • uniplex SW markers to see quality of peaks and range of alleles
  • multiplex SW markers as first glance of how multiplex behaves


Results

  • Frag data: PPTX
  • also using frag data from FHVZ (late November 2012)
    • (apparently, I already did this PCR of uniplexing each SW marker, then throwing it all in together ... oops)


Marker SYR Genotype TP3 Genotype Uniplex comments Multiplex comments
SW614-A1 (NED) 410 415 [poop] tall @ 8000, very clean short @ 300, clean
SW614-A4 (FAM) 395 396 396 398 tall @ 15000 short @ 300
SW614-A6 (PET) [poop] 255 275 tall @ 8000 good @ 1500
SW614-A7 (HEX) 252 252 248 248 tall @ 14000 no amplification
SW614-B1 (HEX) 316 316 299 316 tall @ 16000, one replicate of SYR poop good @ 1200
SW614-B6 (FAM) 308 310 308 310 tall @ 11000 no amplification
SW614-B10 (FAM) 188 194 186 198 tall @ 12000, one replicate of SYR violates slope rule no amplification
SW614-B12 (NED) 285 285 255 274 tall @ 14000 good @ 2000


  • regarding sar80: after reviewing past PCRs using sar80, seems monomorphic in diversity panel, doesn't multiplex well ... no need to use this finicky marker


  • definitely move forward with SW614-A4, SW614-B1 and SW614-B12 as SCOC MP2
  • may consider adding SW614-A6, just to include that color. I can possibly imagine that SYR's genotype is 260 263 (with lots of stutter), which means other samples may be just fine. Might as well include the marker, and if scoring ends up being too difficult, then we'll dump the marker.



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