Arichard:Notebook/2013/September: Difference between revisions

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>Andrew
(Created page with "==September, 2013== ===09/03/2013=== Chris and I will be taking responsibility for MIDAS from here on. Started overnight MDA with Jeff and Chris observing. Loaded 8 arrays ...")
 
>Andrew
Line 9: Line 9:
* 2 conditions:
* 2 conditions:
** 4 arrays with protease (trypsin)
** 4 arrays with protease (trypsin)
** 4 arrays, standard protocol except no freeze thaw
** 4 arrays, [[Arichard:Protocols/MDA on microarray 2013 09 05|standard protocol]] except no freeze thaw


Did not image prior to ALS because chromosomes were expected in every well due to high concentration.
Did not image prior to ALS because chromosomes were expected in every well due to high concentration.
Line 30: Line 30:
*** Total = 11.2 ul
*** Total = 11.2 ul


* No protease condition received 11.2 ul standard MDA master mix
* No protease condition received 11.2 ul [[Arichard:Protocols/MDA on microarray 2013 09 05|standard MDA master mix]]


===09/04/2013===
===09/04/2013===

Revision as of 01:04, 6 September 2013

September, 2013

09/03/2013

Chris and I will be taking responsibility for MIDAS from here on.

Started overnight MDA with Jeff and Chris observing. Loaded 8 arrays with PGP-1 chromosomes.

  • 2 conditions:

Did not image prior to ALS because chromosomes were expected in every well due to high concentration.

  • Modification to standard protocol for protease treatment:
    • Load 3 ul chromosomes. Add coverslip, let sit 10 min @ RT
    • Load 3 ul 0.25% trypsin (1X) into protease condition arrays. Cover and let sit 5 min @ RT
    • Load 4.5 ul ALS into all arrays. Cover and incubate 10 min @ 40 degC in thermocycler with plate adapter
    • Load 4.5 ul NS into all arrays.
      • Protocol based on Quake haplotype paper (Fan et al. 2010 NBT)
  • Protease condition master mix(per rxn/array):
    • 15 ul template
    • 1 ul 1 mM N6*
    • 1 ul 25 mM dNTP (Epicentre)
    • 2.3 ul 10X Phi29 buffer
    • 0.1 ul 50X SYBR Green
    • 1 ul 23X protease inhibitor (cOmplete)
    • 1 ul H2O
      • Total = 11.2 ul

09/04/2013

Extracted and processed 14 samples + 2 NTCs (no extraction)

1 NTC showed large amount of amplification during blue/orange PCR, more than all other samples. Reagent contamination would have been seen in all samples.

  • 2 samples confirmed positive on gel. Nextera indexes #28 and #32.

09/05/2013

Chris started overnight MDA with Jeff and I observing. Loaded 8 arrays with neuronal nuclei.