User:Tara K. Luckau/Notebook/Team ConGen/2013/04/17

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==CNTI Acos5==
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==SCOC MP2 State of the Multiplex==
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===PCR===
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===Summary of Past Results===
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* comments
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* SW614- A4, A6, B1, B12
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:: [[Image:20130110_PCRa.png|800 px]]
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** gelled well
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** amplified well in uniplex
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** amplified well in multiplex with all 8 SW markers
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===Gel===
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* multiplex with A4 + A6 + B1 + B12
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** gel blank
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** frag fail  ?!
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** maybe due to Techne cycler?
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{| {{table}} style="text-align: center"
 
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|-
 
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! Pour !! Load !! Run
 
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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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|-
 
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| 27 µL GelRed || 6 µL ladder || 45 minutes
 
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|}
 
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* re-do of multiplex with A4 + A6 + B1 + B12
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** (didn't run gel)
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** frag failed again!
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** ran on Techne again - thermalcycler poopy?
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:: [[Image:20130110_Gela.png|800 px]]
 
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* re-re-do of multiplex with A4 + A6 + B1 + B12
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** gel blank
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** frag failed AGAIN!
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** ran on Opticon  :(
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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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===Next Steps===
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===Fragment Analysis Submission===
 
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* UAGC Submission# ????
 
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:: [[Image:20130227_Frag.png|900 px]]
 
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* FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151779 801124151779]
 
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:: [[Image:20130227_FedEx.png|700 px]]
 
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* FedEx picked up ____pm, 26 February
 
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* FedEx delivered ____am, 27 February
 
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* UAGC received ____am, 27 February
 
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* UAGC completed ____am, 28 February
 
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* redo it ALL to figure out what the problem is
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* potential problems:
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'''** DNA - run 4 samples, not just the 2 controls'''
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** fluorophore - can re-order labelled primers, but only after testing other options because of price
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** primer interactions - none to be concerned about
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** cycler - avoid using Techne until properly tested
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** 3730 - Crystal reports no problems with other clients' data
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'''** uniplex/4-plex/8-plex - just re-do these reactions'''
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** Taq - can run 2 replicates using 2 different lots of Taq, but don't know what lot I used for the above failures, so not a good test
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** Buffer - don't know how to test

Revision as of 17:41, 9 June 2013

Tara's Lab Notebook Main project page
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Contents


SCOC MP2 State of the Multiplex

Summary of Past Results

  • SW614- A4, A6, B1, B12
    • gelled well
    • amplified well in uniplex
    • amplified well in multiplex with all 8 SW markers


  • multiplex with A4 + A6 + B1 + B12
    • gel blank
    • frag fail  ?!
    • maybe due to Techne cycler?


  • re-do of multiplex with A4 + A6 + B1 + B12
    • (didn't run gel)
    • frag failed again!
    • ran on Techne again - thermalcycler poopy?


  • re-re-do of multiplex with A4 + A6 + B1 + B12
    • gel blank
    • frag failed AGAIN!
    • ran on Opticon  :(


Next Steps

  • redo it ALL to figure out what the problem is
  • potential problems:

** DNA - run 4 samples, not just the 2 controls

    • fluorophore - can re-order labelled primers, but only after testing other options because of price
    • primer interactions - none to be concerned about
    • cycler - avoid using Techne until properly tested
    • 3730 - Crystal reports no problems with other clients' data

** uniplex/4-plex/8-plex - just re-do these reactions

    • Taq - can run 2 replicates using 2 different lots of Taq, but don't know what lot I used for the above failures, so not a good test
    • Buffer - don't know how to test



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