Labeling enzyme digestion to release labeling experiments

Summary

is complementary to the labeling enzyme technique in differential display (DD) experiments

Modern Neuroscience Research Techniques

Author(s): U. Windhorst & H. Johansson Translated by Z. Q. Zhao Jun Chen

Operation method

Labeling enzyme digestion to release labeling experiments

Materials and Instruments

Solutions & Buffers LoTE
Eppendorf Test Tubes

Move

Experimental program A

1. Add the following to each test tube containing Dynabeads:

2. Enzymatic digestion at 65°C for Ih (mixing every IOmin).

3. Collect the supernatant (containing the released cDNA labeling) at the time of

4. Amplify to 200mL with LoTE.

5. Equal volume of PCI extraction, ethanol precipitation (protocol A, step 2).

6. Resuspend the rinsed and air-dried precipitate in 10 ul LoTE.

Experimental program B

1. Remove the ligation reaction mixture from the PCR tube and wash once with 50ul of wash solution.

2. Remove the rinse solution from the tube, add 50 ul of 1X Restriction Buffer, and remove the restriction buffer.

3. Add the following to the test tube:

4. Digest at 65°C for lh.

5. Add 175ul of LoTE and transfer to a 1.5 ml Eppendorf tube. Do not discard as the mixture contains the released SAGE tag attached to the fittings.

6. Extract with an equal volume of PCI as described above and ethanol precipitate (Experimental protocol A, step 2).

7. Resuspend the rinsed and air-dried precipitate in 21.5ul of LoTE.

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