User:Tara K. Luckau/Notebook/Team ConGen/2010/11/08: Difference between revisions

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* 27µL Gel Red (added when agarose turned clear but still hot, used stir bar)  
* 27µL Gel Red (added when agarose turned clear but still hot, used stir bar)  
===Load Gel===
===Load Gel===
* 5µL loading dye + 5µL sample
* 5µL NanoPure H<sub>2</sub>O + 5µL sample
**(to conserve loading dye)
**(don't need loading dye in every sample since it's in ladder)
* 10µL ladder / loading dye mix
* [[Image:20101108 GelSchematic.jpg|800 px]]
* [[Image:20101108 GelSchematic.jpg|800 px]]
===Run Gel===
===Run Gel===
* 130C, 2 hrs
* 130C, 2 hrs
===Image Gel===
===Image Gel===
*  
* UGH!
* [[Image:20101108_Top.TIF|300 px]]    [[Image:20101108_TopZoom.TIF|300 px]]
* [[Image:20101108_Bottom.TIF|300 px]]    [[Image:20101108_BottomZoom.TIF|300 px]]
* 3 faint bands
 
===Results===
===Results===
*  
* Very faint bands at E8, E10, E12, corresponding to DNA concentration of 1x, Buffer G, Temperatures 61, 63, 65
 
* around 150 bp band


==Next Steps==
* there's obviously a problem:
** reagent sucks (made wrong, not stored properly, expired)
** inhibition
* Start weeding out the problem
** check math on dNTPs, ladder (too bright?)
** try different Taq?
** try Scun22 primers
**




Revision as of 18:10, 8 November 2010

Tara's Lab Notebook <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Gel Scun2 F-R with DNA Gradient

Cast Gel

  • 270µL 1x TAE + 5.4g agarose
  • hot plate + stir bar
  • 27µL Gel Red (added when agarose turned clear but still hot, used stir bar)

Load Gel

  • 5µL NanoPure H2O + 5µL sample
    • (to conserve loading dye)
    • (don't need loading dye in every sample since it's in ladder)
  • 10µL ladder / loading dye mix

Run Gel

  • 130C, 2 hrs

Image Gel

  • UGH!
  • 3 faint bands

Results

  • Very faint bands at E8, E10, E12, corresponding to DNA concentration of 1x, Buffer G, Temperatures 61, 63, 65
  • around 150 bp band

Next Steps

  • there's obviously a problem:
    • reagent sucks (made wrong, not stored properly, expired)
    • inhibition
  • Start weeding out the problem
    • check math on dNTPs, ladder (too bright?)
    • try different Taq?
    • try Scun22 primers



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