互联网2013-11-13
Additional materials to be supplied by user
1. RNase-free Lysozyme
2. TE buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA) Procedure
1. Harvest Cells and resuspend in 500 ul TE/lysozyme and incubate at RT for 7 min. Centrifuge 1010 cells at 4,000 x g for 5 min. Discard supernatant and add 100 ul of TE buffer containing lysozyme (0.5 mg/ml for Gram-negative and 4 mg/ml for Gram-positive bacteria). Resuspend cells completely and incubate at room temperature for 7 min.
2. Add 2 ml of TRK Lysis Buffer and mix by pipetting several times. Remember to add 20 ul of ß-mercaptoethanol per 1 ml of TRK Lysis Buffer.
3. Add 1.5 ml 100% ethanol to lysate and mix by vortexing. A precipitate may form at this point. This will not interfere with RNA purification.
4. Apply sample (approximately 3.5 ml) from step 3 to an HiBind® RNA Midispin column. With the column mounted in a clean 15 ml collection tube (supplied with kit) centrifuge 5 min at 5000 x g (at room temperature) in a centrifuge. Discard flow-through and proceed to step 5. (This is the starting point for optional DNase I digestion treatment, see page 6 for protocol)
5. Wash column with 3ml RNA Wash Buffer I. Centrifuge 5 min at maximum speed and discard both flow-through and collecting tube.
6. Place spin column into a 8 ml clean collection tube (supplied) and add 3 ml RNA Wash Buffer II diluted with ethanol. Centrifuge and discard flow-through as above. Reuse the collection tube in step 7.
Note: Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.
7. Wash column with a second 3 ml 1 X Wash Buffer II. Repeat step 6 and discard flow-through. Then empty the collection tube and centrifuge the spin cartridge for 10 min at 4000-5000 x g to completely dry the HiBind™ matrix.
8. RNA Elution. Transfer column to a clean 15 ml collection tube (Not supplied) and elute RNA with 250-500ul DEPC-treated water (supplied with kit). Centrifuge column for 10 min at 4,000-5,000 x g. If the expected RNA yield > 500 ug the a second elution may be required. Elution with two 250 ul aliquots is no more efficient than with one 500 ul aliquot.
All centrifugation steps must be carried out at room temperature.
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2小时能完成Western Blot实验，可能吗？
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