Daniel:Notebook/HiResChrPaint/2013-4-22: Difference between revisions

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  Purpose: Start new probe synthesis (from scratch) to better assess methods.  Getting low yields on dye coupling reaction,
  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
  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.
  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==
==Expansion qPCR==
1.  Set up 8 reactions for each of the 2 probes (16 reactions total)
1.  Set up 8 reactions for each of the 2 probes (16 reactions total)


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
|style="background-color:#FFFF00" align="right" width="149" height="16" | &nbsp;
|style="background-color:#FFFF00" align="right" width="114" height="15" align="right" | &nbsp;
|style="font-weight:bold" width="65" | per rxn
|style="font-weight:bold" width="65" align="center" | per rxn
|style="font-weight:bold" width="65" | V4S1
|style="font-weight:bold" width="65" align="center" | V4S3
|style="font-weight:bold" width="65" | V4S2
|style="font-weight:bold" width="65" align="center" | V6S3
|style="font-weight:bold" width="65" | V4S3
|style="font-weight:bold" width="65" | V4S4


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | 20 nM OligoPool
| height="15" align="center" | 20 nM OligoPool
| align="right" | 0.2
| align="right" align="right" | 0.2
| align="right" | 1.7
| align="right" align="right" | 1.7
| align="right" | 1.7
| align="right" align="right" | 1.7
| align="right" | 1.7
| align="right" | 1.7


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | 2X Kapa SYBR qPCR MM
| height="30" align="center" | 2X Kapa SYBR qPCR MM
| align="right" | 50
| align="right" align="right" | 50
| align="right" | 425
| align="right" align="right" | 425
| align="right" | 425
| align="right" align="right" | 425
| align="right" | 425
| align="right" | 425


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | 100 uM AP1V41U
| height="15" align="center" | 100 uM AP1V41U
| align="right" | 0.4
| align="right" align="right" | 0.4
| align="right" | 3.4
| align="right" align="right" | 3.4
| align="right" | 3.4
| align="right" align="right" | 0
| align="right" | 3.4
| align="right" | 3.4


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | 100 uM AP2V4
| height="15" align="center" | 100 uM AP2V4
| align="right" | 0.4
| align="right" align="right" | 0.4
| align="right" | 3.4
| align="right" align="right" | 3.4
| align="right" | 3.4
| align="right" align="right" | 0
| align="right" | 3.4
| align="right" | 3.4


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | 2 mM aa-dUTP
| height="15" align="center" | 100 uM AP1V61U
| align="right" | 3
| align="right" align="right" | 0.4
| align="right" | 25.5
| align="right" align="right" | 0
| align="right" | 25.5
| align="right" align="right" | 3.4
| align="right" | 25.5
| align="right" | 25.5


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | H20
| height="15" align="center" | 100 uM AP2V6
| align="right" | 46
| align="right" align="right" | 0.4
| align="right" | 391
| align="right" align="right" | 0
| align="right" | 391
| align="right" align="right" | 3.4
| align="right" | 391
| align="right" | 391


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | Total Volume
| height="15" align="center" | 50 mM aa-dUTP
| align="right" | 100
| align="right" align="right" | 1/8
| align="right" | 850
| align="right" align="right" | 1
| align="right" | 850
| align="right" align="right" | 1
| align="right" | 850
| align="right" | 850


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
|style="background-color:#FFFF00" align="right" height="16" | &nbsp;
| height="15" align="center" | H20
|style="font-weight:bold" | per rxn
| align="right" align="right" | 46
|style="font-weight:bold" | V6S1
| align="right" align="right" | 415
|style="font-weight:bold" | V6S2
| align="right" align="right" | 415
|style="font-weight:bold" | V6S3
|style="font-weight:bold" | V6S6


|- style="font-size:12pt" align="center"
|- style="font-size:12pt"
| height="16" | 20 nM OligoPool
| height="15" align="center" | Total Volume
| align="right" | 0.2
| align="right" align="right" | 100
| align="right" | 1.7
| align="right" align="right" | 850
| align="right" | 1.7
| align="right" align="right" | 850
| align="right" | 1.7
| align="right" | 1.7


|- style="font-size:12pt" align="center"
|}
| height="16" | 2X Kapa SYBR qPCR MM
| align="right" | 50
| align="right" | 425
| align="right" | 425
| align="right" | 425
| align="right" | 425


|- style="font-size:12pt" align="center"
  2. qPCR
| height="16" | 100 uM AP1V61U
    95 C 30 seconds
| align="right" | 0.4
    Repeat 16x
| align="right" | 3.4
      95 C 3 seconds
| align="right" | 3.4
      55 C 30 seconds
| align="right" | 3.4
      72 C 20 seconds
| align="right" | 3.4
      Read Plate
    72 C 2 min
    15 C Hold


|- style="font-size:12pt" align="center"
==Ethanol Precipitation==
| height="16" | 100 uM AP2V6
| align="right" | 0.4
| align="right" | 3.4
| align="right" | 3.4
| align="right" | 3.4
| align="right" | 3.4


|- style="font-size:12pt" align="center"
  1.  Pool half samples for given probe set into 1.5 mL eppendorf tube (4 tubes, 400 uL each)
| height="16" | 2 mM aa-dUTP
  2.  To each 1.5 mL eppendorf add:
| align="right" | 3
    1000 uL EtOH (Rec 2.5X)
| align="right" | 25.5
    40 uL 3M NaOAc (Rec 0.1X)
| align="right" | 25.5
    5 uL Glycoblue (Rec 1/300X)
| align="right" | 25.5
  3. Freeze tubes overnight at -80C ([[Daniel:Notebook/HiResChrPaint/2013-4-23|Continued on 4/23/2103]])
| align="right" | 25.5
 
|- style="font-size:12pt" align="center"
| height="16" | H20
| align="right" | 46
| align="right" | 391
| align="right" | 391
| align="right" | 391
| align="right" | 391
 
|- style="font-size:12pt" align="center"
| height="16" | Total Volume
| align="right" | 100
| align="right" | 850
| align="right" | 850
| align="right" | 850
| align="right" | 850
 
|}

Latest revision as of 22:51, 22 April 2013

Probe Prep S3 4/22/2013[edit]

Back to Calendar

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)