For quantitating RNA levels from either polyA+ selected RNA or crude RNA preps.

0.4 M PIPES 12.1 g PIPES (Sigma #P6757)

4.0 M NaCl 23.4 g NaCl

10 mM EDTA 2.0 ml 500 mM EDTA

pH to 6.7 and bring up to 100 ml with DEPC treated Q, autoclave

8 ml formamide

1 ml DEPC treated Q

1 ml 10X Hybridization Buffer

20 mM Tris 7.5 2 ml 1M Tris 7.5

10 mM EDTA 2 ml 500 mM EDTA 8.0

0.6 M NaCl 12 ml 5 M NaCl

up to 100 ml DEPC treated Q,

autoclave

20 g SDS

up to 100 ml with Q

1 liter mili Q water

1 ml DEPC

shake overnight at 37°

autoclave twice

Proteinase K (10mg/ml in Q), RNaseA (2mg/ml), RNaseT1 (100 mg/ml).

• To make the riboprobe linearize the appropriate template (5 mg) with the appropriate enzyme (5' overhang is best). Phenol/chloroform extract, EtOH ppt, wash and dry. Resuspend in 10 ml DEPC treated Q.

6 ml DEPC Q 3 ml DEPC Q

2 ml 10X Pol. Buffer (NEB) 4 ml 5X Pol. Buffer (Promega)

1 ml 10 mM ATP 1 ml 10 mM ATP

1 ml 10 mM GTP 1 ml 10 mM GTP

1 ml 10 mM CTP 1 ml 10 mM CTP

1 ml 0.05 mM UTP 1 ml 0.05 mM UTP

1 ml RNaseIN 1 ml RNaseIN

1 ml Template (0.5 mg) 1 ml Template (0.5 mg)

5 ml a32P UTP (800 Ci/mmol) 5 ml a32P UTP (800 Ci/mmol)

1 ml T7 RNA Polymerase (NEB) 1 ml SP6 RNA Polymerase (Promega)

2 ml 100 mM DTT

• Add 1 ml DPRF DNaseI and incubate at 37°C for 15 minutes, repeat.

• Dry down 5-20 mg RNA (crude) and resuspend in 29 ml 1X Hybridization mix. Add 1 ml riboprobe and incubate at 48°C overnight.

• Make the RNase Mixture and add 380 ml per tube. Incubate for 30 minutes at room temperature.

42 ml 84 ml 126 ml 168 ml RNaseA

42 ml 84 ml 126 ml 168 ml RNaseT1

• Add 10 ml 20% SDS, 10 ml Proteinase K and incubate at 37°C for 15 minutes. Phenol/chloroform extract, and remove 360 ml of the aqueous phase. Add 1 ml EtOH (no salt required) and 1 ml tRNA. Spin, wash and dry.

• Resuspend in 10 ml Formamide Loading Dye and run on a 7-8% sequence gel (see protocol O.1).