Daniel:Notebook/RNAFISH/2015-2-24: Difference between revisions

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(Created page with "=Linnarsson Protocol= Back to Calendar I'll be starting work today on the Linnarsson lab protocol for doing RNA FISH on tissue sections. The pro...")
 
>Djacobse
 
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The protocol uses several solutions that are prepared beforehand and aliquoted for later use. Most of these need to only be done once.
The protocol uses several solutions that are prepared beforehand and aliquoted for later use. Most of these need to only be done once.


*E Coli tRNA 20 mg/mL
*'''E Coli tRNA 20 mg/mL'''
**tRNA 500 mg
**tRNA 100 mg
**RNAse free H2O 12.5 mL
**RNAse free H2O 2.5 mL
**aliquot into 500 uL increments and store at -20C
**aliquot into 500 uL increments and store at -20C
*DAPI stock solution
*'''DAPI stock solution'''
**RNAse free water 250 uL
**RNAse free water 500 uL
**DAPI powder 5 mg
**DAPI powder 10 mg
**aliquot into 20 uL increments and store at -20C
**aliquot into 5 20 uL increments and store at -20C
*DAPI ready to use
**store the remainder at -20C
*'''DAPI ready to use'''
**DAPI stock (20 mg/mL) 10 uL
**DAPI stock (20 mg/mL) 10 uL
**nfH2O 990 uL
**nfH2O 990 uL
**Store at 4C use within 1 month
**Store at 4C use within 1 month
*70% Ethanol
*'''70% Ethanol'''
**700mL absolute EtOH
**700mL absolute EtOH
**300 mL ddi water
**300 mL ddi water
*Paraformaldehyde 4% w/v
*'''Ribonucleoside Vanadyl complex (200mM)'''
**Paraformaldehyde 16% w/v 20 mL
**PBS 10X 8 mL
**RNAse free H2O 52 mL
**aliquot into 15 mL increments and store at -20C for up to 4 months
**thaw the solution with 37C water bath before use
*Formamide
**Bring the bottle at RT and prepare 50mL aliquots;
**store them in the dark at 4C for up to 4 months
**Work in a fume hood
*Ribonucleoside Vanadyl complex (200mM)
**Incubate the purchased solution in a water bath at 65C for 10 minutes
**Incubate the purchased solution in a water bath at 65C for 10 minutes
**Aliquot in 100 uL and store up to 4 months at -20C
**Aliquot in 100 uL and store up to 4 months at -20C
*BSA 50 mg/mL
*'''BSA 50 mg/mL'''
**BSA 500 mg
**Came as 1 mL of 50 mg/mL, not going to split it
**RNAse free water 10 mL
 
**Aliquot in 500 uL and store at -20C up to 4 months
NOTES:
*I need fresh formamide and fresh paraformaldehyde 16% w/v  I will order both of these
*For now, I am using less fresh bottles
 
==Dye Coupling==
 
*FOXP2-488
*GAD1-594
 
#Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
#Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
#Denature samples for 5 minutes at 95C, then snap cool using ice box
#Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
##dye cannot be saved for later use.  Use immediately!
#Add 2 uL dye to sample
#Incubate in the dark for 1 hour
#Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation
##Use TE Buffer
#Ethanol Precipitation; incubate overnight
#Continued [[Daniel:Notebook/RNAFISH/2015-2-25|tomorrow]]

Latest revision as of 18:58, 25 February 2015

Linnarsson Protocol[edit]

Back to Calendar

I'll be starting work today on the Linnarsson lab protocol for doing RNA FISH on tissue sections. The protocol is given to me from Simone, one of their main RNA FISH researchers, who visited our lab in Dec 2014/Jan 2015.

Buffer Prep[edit]

The protocol uses several solutions that are prepared beforehand and aliquoted for later use. Most of these need to only be done once.

  • E Coli tRNA 20 mg/mL
    • tRNA 100 mg
    • RNAse free H2O 2.5 mL
    • aliquot into 500 uL increments and store at -20C
  • DAPI stock solution
    • RNAse free water 500 uL
    • DAPI powder 10 mg
    • aliquot into 5 20 uL increments and store at -20C
    • store the remainder at -20C
  • DAPI ready to use
    • DAPI stock (20 mg/mL) 10 uL
    • nfH2O 990 uL
    • Store at 4C use within 1 month
  • 70% Ethanol
    • 700mL absolute EtOH
    • 300 mL ddi water
  • Ribonucleoside Vanadyl complex (200mM)
    • Incubate the purchased solution in a water bath at 65C for 10 minutes
    • Aliquot in 100 uL and store up to 4 months at -20C
  • BSA 50 mg/mL
    • Came as 1 mL of 50 mg/mL, not going to split it

NOTES:

  • I need fresh formamide and fresh paraformaldehyde 16% w/v I will order both of these
  • For now, I am using less fresh bottles

Dye Coupling[edit]

  • FOXP2-488
  • GAD1-594
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 2 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
  8. Centri-Sep column purification after incubation
    1. Use TE Buffer
  9. Ethanol Precipitation; incubate overnight
  10. Continued tomorrow