Riboprobe preparation for in situ hybridization

Promoter- DNA with same sequence as mRNA needed

Linearize 10ug of plasmid DNA using 10x excess of appropriate enzyme (10U to 1 ug of plasmid DNA) 3h - O/N
Run 1ul on gel to verify complete cutting
Treat with 0.1ug/ul of Proteinase K for 30min at 37C to eliminate RNases and polymerases
Purify with Miniprep column. Elute in 31ul of warm Rnase-free H2O (let stand 1-2min before spinning). Place on ice.
Dilute 1ul of reaction 1:10 with 9ul H2O. Load 1ul and 5ul with 5ul ladder to quantify linear DNA/ul

Use Roche in vitro transcription reagents and assemble the following components in order on ice
1-2 ug of linearized template
10ul Roche 10X DIG UTP mix
10ul Roche 10x transcription buffer
5ul Roche Protector RNase Inhibitor
Make up to 90ul with RNase-free H2O (not DEPC)
Then add 10ul Roche T7 or SP6 or T3 depending on the promoter
Mix, spin down, wrap in parafilm and incubate 37C 2h

Remove tube from water bath, add 10ul Roche Dnase I (10U/ul) and incubate at 37C for 15min to destroy DNA template
Add 4ul 0.5M EDTA to terminate DNase reaction. Keep tubes on ice.

Purify with Roche Quick Spin columns.
Tap off top cap, remove bottom cover and drain fluid
Place column in small colletion tube and place both in 15ml Falcon tube and spin 800g for 2min
Change collection tube to fresh collection tube and load reaction (110ul) to center of column and spin 800g for 4min.
Transfer purified probe to fresh eppies
Add 1/9th volume 3M NaAcetate (RNase-free), pH5.5 (12.2ul for 110ul reaction). Mix well and add 3 vol of ice cold 100% EtOH (RNase-free). Mix well and ppt at -80C 3h-O/N

Spin at 4C max for 30min
A large white pellet should be visible. Remove as much EtOH as possible
Add 700ul cold 75% EtOH (RNase-free) to wash DNA
Spin 4C 15min
Remove as much EtOH as possible, air dry for 1-2min
Add 21ul RNase-free H2O, mix gently, then place tube at 55C with lid open to remove any residual EtOH.
Dilute 1ul probe 1:10 and run 1ul and 5ul and marker on agarose gel. Run fast (150V)

Bring probe in top quality formamide HYB buffer to final concentration of 0.1ug/ul and store -20C for months