User:Tara K. Luckau/Notebook/Team ConGen/2012/03/27: Difference between revisions

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==CNTI Acos5==
==SCOC Multiplex Optimization==




===PCR===
===PCR===
* [[Image:20120110_PCRa.png|800 px]]
* Problem: marker ranges in yellow lane of SCOC Multiplex may be wider than currently set (particularly Scun11)
* Experiment: run Scun3, Scun11 and sar80 in uniplex on wide range of samples to establish marker ranges
* [[Image:20120327_PCRa.png|800 px]]




===Gel===
* Problem: Scun19 showing up low intensity
* Experiment: decrease tall Scun15, increase short Scun19 to even out peak heights
* [[Image:20120327_PCRb.png|800 px]]


{| {{table}} style="text-align: center"
|-
! Pour !! Load !! Run
|-
| 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V
|-
| 27 µL GelRed || 6 µL ladder || 45 minutes
|}


* Problem: sar80 seems to want to be run in uniplex because it doesn't play well with others in the sandbox
* Experiment: try to stick sar80 primers into MPG (same buffer and annealing temperature as MPY) to prevent having to run sar80 in uniplex always
* [[Image:20120327_PCRc.png|800 px]]
==CNHY Multiplex Optimization==
===PCR===
* start trying to group markers together into multiplexes
* start with same volume of each primer (can modify primer volumes based on results of this PCR)
====CNHY MPA====
* Ai5033, Ai5057, Ai5037, Acos5, Ai5035, Ai5062
* [[Image:20120327_PCRd.png|800 px]]
====CNHY MPB====
* Ai5043, Acos3, Ai5013, Ai5071
* [[Image:20120327_PCRe.png|800 px]]


[[Image:20120110_Gela.png|800 px]]


* mispriming everywhere - primer pair not usable for CNHY
* lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions
* favorable conditions indicated by [[Image:greenhappyface.png|25 px]]




Revision as of 12:22, 27 March 2012

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SCOC Multiplex Optimization

PCR

  • Problem: marker ranges in yellow lane of SCOC Multiplex may be wider than currently set (particularly Scun11)
  • Experiment: run Scun3, Scun11 and sar80 in uniplex on wide range of samples to establish marker ranges


  • Problem: Scun19 showing up low intensity
  • Experiment: decrease tall Scun15, increase short Scun19 to even out peak heights


  • Problem: sar80 seems to want to be run in uniplex because it doesn't play well with others in the sandbox
  • Experiment: try to stick sar80 primers into MPG (same buffer and annealing temperature as MPY) to prevent having to run sar80 in uniplex always


CNHY Multiplex Optimization

PCR

  • start trying to group markers together into multiplexes
  • start with same volume of each primer (can modify primer volumes based on results of this PCR)


CNHY MPA

  • Ai5033, Ai5057, Ai5037, Acos5, Ai5035, Ai5062


CNHY MPB

  • Ai5043, Acos3, Ai5013, Ai5071





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