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

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==CNTI Acos5==
==SCOC MP2 State of the Multiplex==




===PCR===
===Summary of Past Results===




* comments
* SW614- A4, A6, B1, B12
:: [[Image:20130110_PCRa.png|800 px]]
** gelled well
** amplified well in uniplex
** amplified well in multiplex with all 8 SW markers




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


{| {{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
|}


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


:: [[Image:20130110_Gela.png|800 px]]


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


* 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]]


===Next Steps===


===Fragment Analysis Submission===
* UAGC Submission# ????
:: [[Image:20130227_Frag.png|900 px]]
* FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151779 801124151779]
:: [[Image:20130227_FedEx.png|700 px]]
* FedEx picked up ____pm, 26 February
* FedEx delivered ____am, 27 February
* UAGC received ____am, 27 February
* UAGC completed ____am, 28 February


* 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




Revision as of 15:41, 9 June 2013

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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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