3.3.1. RNA Preparation from ES Cells (see Note 4)
1. Wash cells grown to confluence in a 25-cm2 flask twice with PBS.
2. Add 1.4 mL of guanidinium lysis buffer/107 cells (approx a confluent 25-cm2 flask) swirl flask for about 30 s and collect lysate.
3. Rinse the required number of SW50.1 centrifuge tubes with DEPC-treated water and dry. Add 3.6 mL of CsCl solution to each tube. Draw lysate through a 23-gage needle several times to shear genomic DNA, and carefully layer on top of the CsCl cushion.
4. Spin at 15,000g in a SW50.1 rotor for 16 h at 20°C.
5. Remove the top 4 mL of the gradient, and wash the sides of the tube twice with guanidinium lysis buffer. Invert tube, and drain off remainder of gradient. Cut the bottom off the tube with a scalpel, and rinse RNA pellet twice with 70% ethanol. RNA pellet should become visible in 70% ethanol.
6. Resuspend pellet in 0.3 mL 8 M urea buffer, transfer to new tube, and vortex occasionally over a period of 1 h.
7. Add 0.1 mL 0.4 M NaCl. Extract sample twice with an equal volume of phenol/ chloroform followed by one chloroform extraction.
8. Precipitate RNA with 2 vol of absolute ethanol and store RNA as a precipitate at -20°C.
9. To determine the concentration of RNA in the sample, vortex to produce a uniform suspension, and spin down 0.1 mL in a microfuge. Wash RNA pellet in 70% etha-nol, and resuspend in 0.1 mL water. The OD260nm of this sample gives a reasonably accurate measure of the concentration of RNA in the precipitated sample.
3.3.2. RNA Dot Blot to Eliminate Intron-Containing Lines
We have observed that a significant proportion of gene trap insertions fail to utilize properly the splice acceptor of the vector and produce fusion transcripts that hybridize to intron sequences of the vector. Inefficient splicing is predicted to occur if the vector inserts either into exons of genes or in non-pol II transcription units. We have characterized two such insertions and found that both occurred within rRNA genes transcribed by RNA pol I (Sleeman and Skarnes, unpublished results). To eliminate these nongene trap events prior to RACE cloning, we use an RNA dot-blot method to detect cell lines producing intron-containing transcripts.
1. Spin down and resuspend 10 |g of RNA in 20 mL of DEPC-treated water. Add 40 mL of formamide, 14 mL of formaldehyde, and 88 mL of NaP buffer.
2. Heat samples to 55°C for 15 min, and apply one-half of each sample to duplicate sets of wells in the dot-blot apparatus. Leave for 30 min before applying a vacuum.
3. To assemble the dot-blot apparatus, and cut a piece of Hybond N membrane and a piece of Whatman paper to fit the dot-blot apparatus. Prewet the membrane in water, and then soak it in 0.5 M NaP buffer for 10 min. Prewet the Whatman in 0.5 M NaP buffer, and place it underneath the membrane.
4. Draw samples through with a gentle vacuum, and wash wells once with 0.5 M NaP buffer. Disassemble apparatus, remove membrane with paper attached, and crosslink RNA to membrane using Stratalinker (Stratagene) set on autocrosslink. Probe duplicate sets of membrane with intron and reporter gene probes. Results from a typical dot-blot experiment are shown in Fig. 1.
lacZ probe intron probe
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