Daniel:Notebook/HiResChrPaint/2013-4-22

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Probe Prep S3 4/22/2013[edit]

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Purpose: Start new probe synthesis (from scratch) to better assess methods.  Getting low yields on dye coupling reaction,
but I think that the reason is I have much lower yields than Noi's original run.  Going to try the entire synthesis, focusing on only
2 sets, V4S3 and V6S3.  This way I can focus my attention better and use a more appropriately sized trial run.  S3 was chosen because of
its high yield after previous PCRs.  At the end of this run I plan on painting the cells using only this set, again as a trial before the
multi-colored painting.

Expansion qPCR[edit]

1. Set up 8 reactions for each of the 2 probes (16 reactions total)

  per rxn V4S3 V6S3
20 nM OligoPool 0.2 1.7 1.7
2X Kapa SYBR qPCR MM 50 425 425
100 uM AP1V41U 0.4 3.4 0
100 uM AP2V4 0.4 3.4 0
100 uM AP1V61U 0.4 0 3.4
100 uM AP2V6 0.4 0 3.4
50 mM aa-dUTP 1/8 1 1
H20 46 415 415
Total Volume 100 850 850
 2. qPCR
    95 C 30 seconds
    Repeat 16x
      95 C 3 seconds
      55 C 30 seconds
      72 C 20 seconds
      Read Plate
    72 C 2 min
    15 C Hold

Ethanol Precipitation[edit]

 1.  Pool half samples for given probe set into 1.5 mL eppendorf tube (4 tubes, 400 uL each)
 2.  To each 1.5 mL eppendorf add:
    1000 uL EtOH (Rec 2.5X)
    40 uL 3M NaOAc (Rec 0.1X)
    5 uL Glycoblue (Rec 1/300X)
 3.  Freeze tubes overnight at -80C (Continued on 4/23/2103)