Ribonuclea Protection Assay:
Making RNA probe
Linearized DNA (transcribed 3'-5')
•
• Reaction for RNA probeaisles
o 32P-CTP 1mCi/25µl total per order
o aliquot: 2.5 µl, 3.7 µl, 5.0 µl (commonly using 3.7 µl)
o Note: fill out form for Isotope.
1. Mix all reagents in isotope aliquot tube (using microtubes)
Rx'n 10
µl 15 µl* 20 µl add order stored place
callingorigial -20
5.0
Isotope 2.5
3.7
Mix-C(A,G,T) 2.0 3.0 5.0 first -20
5x buffer 2.0 3.0 4.0 first -20
1.2
first -20 DTT 0.6
0.9
RNasin(antiRNa) 0.3 0.4 0.6 cond -20
2.0
third -20
1.5
DNA 1.0
H2O(RNa free) 0.6 1.0 0.2 first -20
-20 Enzyme* 1.0 1.5 2.0 last
* different reaction us different enzyme--for rat OT mRNA, using SP6
RNA polymera
The following protocol is for 15 µl reactionhttp www you jizz
2. Incubate in 37 C incubator for 45-60 min (For Northern blot assay,
incubate 1.5-2 h)
Start to prepare the gel
3. Add 0.6 µl rRNasin and 6.0 µl DNa I
Incubate 37 C incubator for 20min
4. Add 100 µl TE, 150 µl mixture of phenol/chloroform (stored in frig), 4 µl tRNA(stored in -20 C)
5. Vortex and spin 5 min
6. Take top aqueous layer to a clean tube
7. Add 50 µl 10M Am Acetate, 50 µl TE and 800 µl EtOH
8. Incubate in -20 C or -70 C or dry ice for 30 min
(can be kept in -20 C for overnight)
debrandBefore starting any reaction, boil water
9. Centrifuge 15 min at RT
10. Take all the liquid out using tip and napkin
Dry pellet in air for 5-10 min
11. Add 3 µl TE and mix with pipe twice and leave tip in the tube
Add 16 µl loading buffer and mix well
12. Boil the samples for 5 min and remove to ice immediately
13. Quick spin
14. Load to gel
Preparation and running polyacrylamide-gel for probe purification: Pour 100 ml acrylamide gel in flask with vacuum outlet
•
• • • • • • • • • • • • • • Vacuum for 30 min玩笑的英文
During acrylamide vacuum, clean the glass using Contrad, water, and EtOH
Put in spacers and tape at two sides and bottom
Tight sides with clamps
Add 300 µl Ammonium persulfate (AP)(stored in frig) and 60 µl TEMED (stored in frig, brown bottle)
Pour into plate t immediately and put the comb on (don't put in too deep)
Set about 1-2 hr
If the gel is not for same day experiment, cover wet paper towel and Saranwrap to protect from dry (leave on bench for next day)
Boiling water and turn on 37 C Shaking water bath (on radioactive area) Check or make the running buffer from 10x TBE buffer
1 L of 1x TBE: 100 ml 10x TBE + 900 ml dd H2O
Fill in the top and bottom of gel cells up to edge of glass
Tight glass plates and gel cast with clamps in both sides
Take out the comb and wash the wells veral times
Prerun 20-30 min
Load samples after boiled and run at 350 v for 1-2 hrs
Carefully take one glass from gel (starting on the corner)
Keep big glass on the bottom and put the saran wrap on the top of the gel
Isolation of probes
Preparing materials:
Labeled microcentrifuge tubes (according to number of samples) Surgical blade (type:No.11)
Marker
Small size film
Protecting
glass
Paper
trawl
Put film on top of gel, confirm by left hand, and mark top and right edges of film on the gel
•
• • • • • • • • • •
• Keep film on the gel for few conds
what makes it toMake a mark by folding a corner for orientation and develop film
Cut a window on band site of film
Fit the marks on gel and cut band through the window and save them into tubes
divisionalMake another picture to make sure that the band cut completely
nicholas negroponteAdd 400 µl Gel Eluting buffer (gel buffer-stored in frig
Incubate in 37 C shaking water bath for 2 hrs (150-200 bas) or 31/2 -4 hrs (>400 bas)
Transfer supernatant to clean tubes and avoid gel pieces
Add 1 ml EtOH
Incubate on dry ice for 20-30 min
(can be in –80 C freezer up to t 3 hrs)
Starting prepare the RNA samples美丽人生主题曲
Spin the RNA probe for15 min
Continue prepare the RNA samples
Air dry pellets (t upside down and absorb the liquid on wall using •
napkin)
• Make Hybridization buffer (volume of buffer depending on the number of samples)
500 µl buffer:
µl 5x salt (stored in –70 C)
100
µl formamide (stored in frig, working in chemical hood)
400
Make working concentration of probe solution:
•
o add 3 µl TE, and add 50 µl Hybridization buffer to probe tube
o mix well
o take 2 µl to scintillation vial with 2 ml scintiSafe Plus (Fisher Sci) to count
o <10 µg RNA: 0.5-1 million cpm per sample
headers
< 50 µg RNA: 2-3 million cpm per sample
100 µg - 200 µg RNA: 10 million cpm per sample
o counted number / 2 = count per µl
o cpm/ml x total volume in probe tube = total count per purification
