User:Tara K. Luckau/Notebook/Team ConGen/2012/11/27

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(Autocreate 2012/11/27 Entry for User:Tara_K._Luckau/Notebook/Team_ConGen)
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==CNTI Acos5==
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==CNHY MP1a/b Optimization==
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* optimize for marker peak height - vary each primer (based on results from 23 July 2012)
 +
* CNHY MP1a: increase Ai5013, Ai5043, Ai5071
 +
* CNHY MP1a: decrease Acos5
 +
* CNHY MP1b: increase Ai5057, Ai5062
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===PCR===
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===Frag Results===
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* [[Image:20120110_PCRa.png|800 px]]
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* UAGC submission# YFPQ
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===Gel===
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====MP1a====
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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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! !! 23 July 2012 !! this time
|-
|-
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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: blue" | Ai5043 || did not amplify || did not amplify; '''remove from multiplex'''
|-
|-
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| 27 µL GelRed || 6 µL ladder || 45 minutes
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| style="background: green" | Acos3 || amplified well; no changes needed || still fine, though seems to decrease with increasing Acos5 & Ai5071
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|-
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| style="background: yellow" | Acos5 || amplified with high intensity, pull down a bit || 0.04 = too low; 0.06 = inconclusive (failures); 0.08 = too high
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|-
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| style="background: yellow" | Ai5013 || amplified with very low intensity; increase concentration || second peak never got above 250, regardless of concentration; but in Acos5 assay, noticed second peak reached 400 intensity as Acos5 decreased
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|-
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| style="background: red" | Ai5071 || amplified with lower intensity; increase a bit || best at 0.24, 0.28, 0.32 (200-300 intensity only)
|}
|}
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[[Image:20120110_Gela.png|800 px]]
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* since removing the only marker labelled with FAM, move one of the NEDs to FAM
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* NEDs: Acos5 and Ai5013 ... since Ai5013 tends to be low intensity, would be a good one to move to brighter FAM
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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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====MP1b====
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 +
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{| {{table}} style="text-align: center"
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|-
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! !! 23 July 2012 !! this time
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|-
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| style="background: blue" | Ai5033 || amplified well; no changes needed || still fine (no amp for any markers in Ai5057 assay)
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|-
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| style="background: blue" | Ai5057 || did not amplify || did not amplify; '''remove from multiplex'''
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|-
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| style="background: green" | Ai5037 || amplified well; no changes needed || still fine (no amp for any markers in Ai5057 assay)
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|-
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| style="background: yellow" | Ai5035 || amplified well; no changes needed || a bit low in Ai5062 assay (no amp for any markers in Ai5057 assay)
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|-
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| style="background: red" | Ai5062 || amplified but with very low intensity; increase concentration || best at 0.48 (400 & 6000 intensity)
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|}

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Contents


CNHY MP1a/b Optimization

  • optimize for marker peak height - vary each primer (based on results from 23 July 2012)
  • CNHY MP1a: increase Ai5013, Ai5043, Ai5071
  • CNHY MP1a: decrease Acos5
  • CNHY MP1b: increase Ai5057, Ai5062


Frag Results

  • UAGC submission# YFPQ


MP1a

23 July 2012 this time
Ai5043 did not amplify did not amplify; remove from multiplex
Acos3 amplified well; no changes needed still fine, though seems to decrease with increasing Acos5 & Ai5071
Acos5 amplified with high intensity, pull down a bit 0.04 = too low; 0.06 = inconclusive (failures); 0.08 = too high
Ai5013 amplified with very low intensity; increase concentration second peak never got above 250, regardless of concentration; but in Acos5 assay, noticed second peak reached 400 intensity as Acos5 decreased
Ai5071 amplified with lower intensity; increase a bit best at 0.24, 0.28, 0.32 (200-300 intensity only)


  • since removing the only marker labelled with FAM, move one of the NEDs to FAM
  • NEDs: Acos5 and Ai5013 ... since Ai5013 tends to be low intensity, would be a good one to move to brighter FAM


MP1b

23 July 2012 this time
Ai5033 amplified well; no changes needed still fine (no amp for any markers in Ai5057 assay)
Ai5057 did not amplify did not amplify; remove from multiplex
Ai5037 amplified well; no changes needed still fine (no amp for any markers in Ai5057 assay)
Ai5035 amplified well; no changes needed a bit low in Ai5062 assay (no amp for any markers in Ai5057 assay)
Ai5062 amplified but with very low intensity; increase concentration best at 0.48 (400 & 6000 intensity)



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