Daniel:Notebook/RNAFACS/2015-4-11: Difference between revisions

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#Dry the pellet in the hood  
#Dry the pellet in the hood  
#Resuspend pellet in 12 uL TE
#Resuspend pellet in 12 uL TE
===Labeling Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold"
| width="96" height="30"  valign="bottom" | Probe
| width="65" align="center" | ng/uL ssDNA
| width="65" align="center" | pmol/uL dye
| width="85" align="center" | total DNA (ug)
| width="65" align="center" | dye/probe
| width="65" align="center" | bp:dye ratio
| width="85" align="center" | DNA Yield (%)
|- style="font-size:12pt"
| height="15"  valign="bottom" | GAD1-488
| align="center" align="center" valign="bottom" | 96.4
| align="center" align="center" valign="bottom" | 16.0
| align="center" align="center" valign="bottom" | 1.4
| align="center" align="center" valign="bottom" | 1.1
| align="center" align="center" valign="bottom" | 18
| align="center" align="center" valign="bottom" | 64.84
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | RBFOX3-546
| align="center" align="center" valign="bottom" | 70.0
| align="center" align="center" valign="bottom" | 10.5
| align="center" align="center" valign="bottom" | 1.1
| align="center" align="center" valign="bottom" | 1.0
| align="center" align="center" valign="bottom" | 20
| align="center" align="center" valign="bottom" | 50.00
|- style="font-size:12pt"
| height="15"  valign="bottom" | FOXP2-594
| align="center" align="center" valign="bottom" | 30.3
| align="center" align="center" valign="bottom" | 5.5
| align="center" align="center" valign="bottom" | 1.1
| align="center" align="center" valign="bottom" | 1.2
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 21.87
|}


==Hybridization==
==Hybridization==


#Resuspend cells in RPHB with labeled probes (0.5-1ng/uL)
#Pellet nuclei at 1000xg at 4C for 5 minutes; remove supernatant
#Resuspend nuclei in 150 uL RPHB  
#Split nuclei into 3 tubes (50 uL each; ~150K nuclei/tube)
#Add 1 uL labeled probes to 0.5-1ng/uL
#Incubate overnight at 30C
#Incubate overnight at 30C

Latest revision as of 23:00, 11 April 2015

In Tube FISH Test (Started 4/10/2015)[edit]

Back to Calendar

Dye Labeling[edit]

  1. Centrifuge at 4 C for 30 minutes (14000 rpm)
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge at minutes at 4 C
  4. Dry the pellet in the hood
  5. Resuspend pellet in 12 uL TE

Labeling Results[edit]

Probe ng/uL ssDNA pmol/uL dye total DNA (ug) dye/probe bp:dye ratio DNA Yield (%)
GAD1-488 96.4 16.0 1.4 1.1 18 64.84
RBFOX3-546 70.0 10.5 1.1 1.0 20 50.00
FOXP2-594 30.3 5.5 1.1 1.2 17 21.87

Hybridization[edit]

  1. Pellet nuclei at 1000xg at 4C for 5 minutes; remove supernatant
  2. Resuspend nuclei in 150 uL RPHB
  3. Split nuclei into 3 tubes (50 uL each; ~150K nuclei/tube)
  4. Add 1 uL labeled probes to 0.5-1ng/uL
  5. Incubate overnight at 30C