16. 10 mM Tris–HCl pH 8.
17. 6Â Protein sample buffer: 300 mM Tris–HCl pH 6.8, 30%
glycerol, 6% SDS, 600 mM DTT, 0.01% bromophenol blue.
18. 3Â Protein sample buffer supplemented with 2 mM biotin and
20 mM DTT.
19. DMSO.
2.4 On-Beads
Trypsinization (See
Note 2)
1. Iodoacetamide, single use.
2. Dithiothreitol (DTT), No-Weigh™ Format.
3. Sequencing-grade modified trypsin.
4. 250 mM NH 4 HCO 3 stock solution.
2.5 RNA
Manipulations
1. RNase T1 buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 2 mM
EDTA, 1% NP40.
2. RNase T1.
3. Dephosphorylation buffer (based on the calf intestinal phosphatase buffer): 50 mM Potassium acetate, 20 mM Trisacetate, 10 mM magnesium acetate, 0.1 mg/ml BSA,
pH 7.9. Make a 10Â stock.
4. Calf intestinal phosphatase.
5. T4 polynucleotide kinase (PNK) buffer without DTT: 70 mM
Tris–HCl, 10 mM MgCl 2 , pH 7.6.
6. PNK buffer with DTT: 70 mM Tris–HCl, 10 mM MgCl 2 ,
5 mM DTT, pH 7.6. Prepare a 10Â stock.
7. PNK.
8. γ32
P-ATP 10 mCi/mL, 1.6 μM.
9. Proteinase K buffer: 50 mM Tris pH 7.5, 75 mM NaCl,
6.25 mM EDTA, 1% SDS.
10. Proteinase K.
11. 10 mg/mL Glycogen or a stained glycogen product.
12. Acidic phenol-chloroform (pH 4.5).
13. Water-saturated chloroform.
14. Nucleic acid low-binding 1.5 mL tubes.
15. Truncated and mutated RNA ligase 2, T4 Rnl2(1-249)K227Q
(1 mg/mL) (NEB).
16. T4 RNA ligase, T4 Rnl1 (1 mg/mL).
17. 50% DMSO.
18. Low-melting-point agarose.
19. SuperScript™ Reverse Transcriptase (III or IV).
20. Taq DNA polymerase.
Proximity-CLIP
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