Granato Laboratoy
Laura Carlson
Modified 2/9/2009
Making Probe for in situs
SOLUTIONS
PCI (Phenol-chloroform-isoamyl alcohol)
50ul phenol
48ul chloroform
2ul isoamylalcohol
CI (Chloroform-Isoamylalcohol)
96ul chloroform
4ul isoamylalcohol
(make up a stock of CI, store at -20, and add P at the last minute in the reaction tube)
3M NaAc pH 5.6
20.4g NaAc Trihydride
50ml DEPC H2O
pH to 5.6
500mM EDTA
FW 372.2 g/mol
9.3gg in 50ml DEPC H2O
pH to 8.0 using NaOH
Use vigorous stirring, moderate heat and time
1M Tris-Cl pH 7.5
FW 121.4 g/mol
6.05gg in 50ml DEPC H2O
pH to 7.5 using HCl
TE
500ul 1M Tris-Cl pH 7.5
100ul 500mM EDTA pH 8.0
DEPC to 50ml
PROCEDURE
Digestion
1. Digest 5-10ug of plasmid:
6ul 10X buffer
Xul plasmid (5-10ug)
4ul enzyme
H20 to 60ul
2. Incubate for 2hrs at 37°C
3. Take 2ul to run 1% agarose gel
Phenol extraction
1. Add 40ul H2O for a total volume of 100ul
2. Add 1 volume PCI, vortex and centrifuge 1min
3. Transfer upper phase in a new tube and add 1 volume CI, vortex and centrifuge
1min
4. Transfer upper phase to new tube
5. Add 1/10 volume 3M NaAc pH 5.6 and 2 volumes 100% EtOH, vortex 10s
6. Let precipitate 1 hour to ON at -20°C
7. Centrifuge for 10-15min, 15000RPM
8. Wash pellet once in 70% EtOH (DEPC H2O)
9. Centrifuge for 10-15 min
10. Air dry the pellet for 10 min (maybe longer)
11. Add 10.5ul DEPC-TE. Run 0.5ul on a gel to figure out concentration
Transcription
1. Set up:
11ul DEPC H2O + plasmid (1ug)
4ul 5X Transcription buffer (comes with RNA polymerases, see below)
2ul NTP labeling mixture (Roche, DIG RNA labeling mix, Cat# 13037722)
2ul RNA Polymerase (Promega Cat# P207B)
1ul RNAse inhibitor (included in kit)
2. Incubate at 37°C for 2 hrs
3. Take 1ul to run gel
4. Add 1ul DNase, 37°C, 15min optional
5. Add :
3.7ul DEPC H2O
1.33ul 7.5M LiCl
75ul EtOH
6. Precipitate at -20°C O/N
7. Centrifuge 4°C, 13000 RPM for 30 min
8. Wash pellet in 500ul 70% DEPC-EtOH (-20°C)
9. Centrifuge 5 min, 13000 RPM, RT
10. Air dry 5-10 min
11. Add :
100ul DEPC H2O
20 units RNasin (0.7ul)
12. Take 5ul to run gel
13. Store RNA probe at -20°C