Matt:LabNotes/2015-6-15: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "==Motor Neurons RNA FISH Dye Coupling== *Matt:LabNotes/2015-6-8#DARTFISH ===Dye Coupling=== [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coup...") |
>Mzcai mNo edit summary |
||
Line 1: | Line 1: | ||
==Motor Neurons RNA FISH Dye Coupling== | ==Motor Neurons RNA FISH Dye Coupling== | ||
*[[Matt:LabNotes/2015-6-8# | *[[Matt:LabNotes/2015-6-8#RNA_FISH | Previous RNA FISH attempt detected no signal]] | ||
**Try again with higher expressed genes (CUX2 and KIT) | |||
===Probe Resuspension=== | |||
**[[Media:GAO_022315.xlsx | Table of ordered plates]] | |||
***KIT: Plate=KIT-SNAP25-20150128 Wells=A1-D12 | |||
***SNAP25: Plate=KIT-SNAP25-20150128 Wells=E1-H12 | |||
#Resuspend each oligo in 15 uL nuclase-free H2O (1 mM) | |||
#Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM) | |||
##Store the remaining oligos at -20C | |||
#Add 450 uL nuclase-free, diluting 10:1, for final concentration of 100 uM | |||
#*Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower... | |||
#*Goal is to get 1ug/ul DNA concentration | |||
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy<!-- | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''measured (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''normalized (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Average''' | |||
| align="center" style="background:#f0f0f0;"|'''Stdev''' | |||
|- | |||
| KIT 1:1||1228||1228|||| | |||
|- | |||
| KIT 1:5||271.8||1359|||| | |||
|- | |||
| KIT 1:10||135.4||1354||1313.7||74.2 | |||
|- | |||
| SNAP25 1:1||1227.6||1227.6|||| | |||
|- | |||
| SNAP25 1:5||258.5||1292.5|||| | |||
|- | |||
| SNAP25 1:10||128.8||1288||1269.4||36.2 | |||
|- | |||
|} | |||
*For ADARB2 & CUX2 with approx 1300ng/ul | |||
**Add 132ul H2O to 440ul | |||
**1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added | |||
===Dye Coupling=== | ===Dye Coupling=== | ||
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> | [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> |
Revision as of 00:09, 16 June 2015
Motor Neurons RNA FISH Dye Coupling
- Previous RNA FISH attempt detected no signal
- Try again with higher expressed genes (CUX2 and KIT)
Probe Resuspension
- Table of ordered plates
- KIT: Plate=KIT-SNAP25-20150128 Wells=A1-D12
- SNAP25: Plate=KIT-SNAP25-20150128 Wells=E1-H12
- Table of ordered plates
- Resuspend each oligo in 15 uL nuclase-free H2O (1 mM)
- Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
- Store the remaining oligos at -20C
- Add 450 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
- Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
- Goal is to get 1ug/ul DNA concentration
- Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy