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DNA实验

RNase Protection Assay (Bowtell Lab)

2024-10-17 DNA实验 加入收藏
1. Cut 10ug of plasmid DNA where you want the transcript to end.2. Add an equal

1. Cut 10ug of plasmid DNA where you want the transcript to end.

2. Add an equal volume of 2X PK buffer:

200mM TrisHCl pH 7.5

25mM EDTA

300mM NaCl

2% SDS

200ug/ml proteinase K

3. 37� for 30min and then extract with phenol/CHCl3 and ethanol precipitate. Resuspend at 1mg/ml in DDW.

4. Labeling reaction:

2ul 5X SP6 buffer: 200mM TrisHCl, 30mM MgCl2, 10mM spermadine

0.5ul RNAsin

0.5ul 10mM rATP

0.5ul 10mM rCTP

0.5ul 10mM rGTP

5ul a-P32-UTP (400Ci/mMol)

0.5ul template

0.5ul 200mM DTT

0.5ul appropriate RNA polymerase

Incubate for 90-120min at 41�.

5. Add 0.5ul RNAsin and 0.5ul RNAse free DNAse (5mg/ml). Incubate for 15min at 37�.

6. Add 10ug tRNA and 100ul DDW. Phenol/CHCl3 extract and precipitate with 50ul 5M NH4 acetate, 400ul ethanol.

7. Resuspend in 100ul 1X hybridization buffer:

0.1 volume 10X hyb. (400mM pipes, pH 6.4, 4M NaCl, 10mM EDTA)

0.1 vloume DDW

0.8 volume deionized formamide.

1ul should be 1000-2000 cps on the mini monitor.

8. Spin down RNA as an ethanol ppte and resuspend in 24ul 1X hyb. buffer and add 0.5-1.0ul of probe (from step 7). Include a control RNA eg. tRNA or negative tissue.

9. Incubate overnight at 37?50�.

10. Add 350ul RNAse buffer to hybridization and vortex immediately.

1X RNAse buffer:

10mM Tris pH 7.5

5mM EDTA

300mM NaCl

40ug/ml RNAse A (Sigma)

2ug/ml RNAse T1 (Sigma)

11. Incubate at ___ � for ____min.

Optimal times and temperatures vary for different probes. Overdigestion gives lots of smlall bands and underdigestion gives large artifactual bands. 37� for 10-15min works well in many cases.

12. To stop the reaction add 20ul 10% SDS and 5ul 20mg/ml proteinase K. Incubate for 15min 37�.

13. Add 2-10ug tRNA carrier. Phenol chloroform extract and ethanol preciptate without additional salt. Air dry pellet and resuspend in 3-4ul sequencing loading buffer and run on a sequencing gel after boiling.


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