User:Tara K. Luckau/Notebook/Team ConGen/2012/01/06: Difference between revisions

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==CNHY Acos5==
==ASHY Multiplex==




===PCR===
* prepping to start multiplexing CNHY primers ... so see primer interactions ...
* [[Image:20111023_PCRa.png|800 px]]




===Gel===
===Gradient Summary===


{| {{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
|}


* green cells indicate clean amplification; orange cells indicate less-than-ideal conditions, but useable if necessary
* [[Image:20120106 GradientSummary.png]]


[[Image:20111023_Gel.png|800 px]]


* mispriming everywhere - primer pair not usable for CNHY
===Multiplex Map===
* 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]]
 
* markers placed arbitrarily
* marker ranges indicated as in Crawford et al 2007, but approximate amplicon size may be given in final parentheses if Crawford designation is wide or very different from gel.
* [[Image:20120106 Map.png]]
 
 
===AutoDimer===
 
 
* color families (red, green, blue, gray) indicate possible multiplexing based on primer interactions
* [[Image:20120106_AutoDimer.png|800 px]]
 
 
===PCR - determine approximate range of each marker===
 
 
* pool high-quant DNA samples from throughout SD county:
:-- CNHY CPN 02
:-- CNHY LOM 08
:-- CNHY RAJ 01
:-- CNHY RAJ 35
:-- CNHY RAJ 42
:-- CNHY RAJ 50
:-- CNHY TP1 11
:-- CNHY TP3 05
:-- CNHY TP3 19
:-- CNHY TP3 20
* 2µL each DNA STK + 80µL LowTE (to dilute to ~10ng/µL) + 20µL CNHY 062911TKLH ('play' DNA, already 10 ng/µL)
 
 
* [[Image:20120106_PCR.png|800 px]]
 




Revision as of 09:40, 20 February 2013

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ASHY Multiplex

  • prepping to start multiplexing CNHY primers ... so see primer interactions ...


Gradient Summary

  • green cells indicate clean amplification; orange cells indicate less-than-ideal conditions, but useable if necessary


Multiplex Map

  • markers placed arbitrarily
  • marker ranges indicated as in Crawford et al 2007, but approximate amplicon size may be given in final parentheses if Crawford designation is wide or very different from gel.


AutoDimer

  • color families (red, green, blue, gray) indicate possible multiplexing based on primer interactions


PCR - determine approximate range of each marker

  • pool high-quant DNA samples from throughout SD county:
-- CNHY CPN 02
-- CNHY LOM 08
-- CNHY RAJ 01
-- CNHY RAJ 35
-- CNHY RAJ 42
-- CNHY RAJ 50
-- CNHY TP1 11
-- CNHY TP3 05
-- CNHY TP3 19
-- CNHY TP3 20
  • 2µL each DNA STK + 80µL LowTE (to dilute to ~10ng/µL) + 20µL CNHY 062911TKLH ('play' DNA, already 10 ng/µL)





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