Daniel:Notebook/RNAFACS/2015-12-1: Difference between revisions

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>Djacobse
>Djacobse
 
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[[Daniel:Notebook/RNAFACS|Back to Calendar]]
[[Daniel:Notebook/RNAFACS|Back to Calendar]]


Time to test these gels on the slides.  I'm going to test the protocol with and without nuclear lysis.
Samples Matrix
I'll be using samples [[Daniel:Notebook/RNAFACS/2015-11-24|1-1 and 1-2]].
==Protocol==
===Lysis===
This protocol follows Andrew's protocols for:
*[[Arichard:Reagents/als|Alkaline Lysis Solution (ALS)]]
*[[Arichard:Reagents/ns|Neutralization Solution (NS)]]
*[[Arichard:Protocols/MDA on microarray#Lysis|Nuclear lysis]]
For slide 1-1, do the following (in the hood)
#Remove from -80 and leave on ice block
#Add 10 uL ALS to sample; swirl to mix; incubate 10 minutes on ice block
#Add 10 uL NS; swirl to mix
#Flick off neutralized solution
===RNA Scope===
#Remove slide 1-2 from freezer, add 20 uL 4% paraformaldehyde in PBS to each slide
#Rinse slides in 1X PBS; repeat
#Dehydration
##Incubate slides in 50% EtOH for 5 minutes at RT
##Incubate slides in 70% EtOH for 5 minutes at RT
##Incubate slides in 100% EtOH for 5 minutes at RT
##Incubate slides in 100% EtOH for 10 minutes at RT
#Rehydration
##Incubate slides in 70% EtOH for 2 minutes
##Incubate slides in 50% EtOH for 2 minutes
##Incubate slides in 1X PBS for 10 minutes
#Apply Pretreat 3
##Remove excess liquid; add 2-4 drops of pretreat 3
##Incubate for 10 min at RT (in oven)
##Flick to remove excess liquid
##Rinse in fresh 1X PBS; be sure to agitate to clean well
===Multiplex Fluorescent Assay===
#Prepare-Reagents
##Prepare probes by warming to 40C for 10 minutes, then cool to RT
##Briefly spin down C2 and C3 probes to collect the liquid
##Combine C2, C3, and C1 probes by pipetting into a new 1.5mL tube at 1:1:50 ratio
##Prepare 1X Wash Buffer
##Place AMP1-4 FL reagents at RT
##Ensure HybEZ OVEN and Control Tray are at 40C
#Hybridize Probe
##Flick to remove excess liquid; add 2-4 drops of probe
##Incubate for 2 hrs at 40C
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
#AMP1-FL
##Flick to remove excess liquid
##Add 2-4 drops of '''AMP1-FL'''; Incubate for 30 minutes at 40C
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
#AMP2-FL
##Flick to remove excess liquid
##Add 2-4 drops of '''AMP2-FL'''; Incubate for 15 minutes at 40C
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
#AMP3-FL
##Flick to remove excess liquid
##Add 2-4 drops of '''AMP3-FL'''; Incubate for 30 minutes at 40C
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
#AMP4-FL
##Flick to remove excess liquid
##Add 2-4 drops of '''AMP4-FL ALT B'''; Incubate for 15 minutes at 40C
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
#Counterstain and Mount
##Flick to remove excess liquid;
##Add 2-4 drops '''DAPI'''; Incubate 30 sec at RT
##Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium
##Seal with a coverslip; avoid bubbles
#Store in the dark at 4C
#Recommended Viewing at 20-40X magnification


Didn't have time today to start testing, so I'll do it [[Daniel:Notebook/RNAFACS/2015-12-2|tomorrow]].  Today I prepped fresh ALS and NS and tested against each other.  From now on, I'll use the below as my formula.
|
===Buffers===
===Buffers===
*Alkaline Lysis Solution (ALS)
*Alkaline Lysis Solution (ALS)
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**2 ul 0.5 M EDTA
**2 ul 0.5 M EDTA
*Neutralization Solution (NS)
*Neutralization Solution (NS)
**3 mL H20
**65 uL H20
**3 mL 2M Tris-HCl
**15 uL 4M Tris-HCl
**4 mL 1N HCl
**20 uL 2N HCl

Latest revision as of 16:53, 2 December 2015

PA Gel Slide Testing[edit]

Back to Calendar


Didn't have time today to start testing, so I'll do it tomorrow. Today I prepped fresh ALS and NS and tested against each other. From now on, I'll use the below as my formula. |

Buffers[edit]

  • Alkaline Lysis Solution (ALS)
    • 80 ul Nuclease free H2O.
    • 8 ul 5 M KOH.
    • 10 ul 1 M DTT.
    • 2 ul 0.5 M EDTA
  • Neutralization Solution (NS)
    • 65 uL H20
    • 15 uL 4M Tris-HCl
    • 20 uL 2N HCl