Anchoring enzyme digestion of cDNA

Summary

If the previous step in the series of analytical techniques for gene expression, SAGE cDNA, was stored at -20°C, then this experiment was thawed slowly on ice.

Modern Neuroscience Research Techniques

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

Operation method

Anchoring enzyme digestion cDNA assay

Materials and Instruments

Solutions & Buffers Primers
Kit MPC-E PCR Instrument Gene PulserII System

Move

Experimental program A

1. Digest the double-stranded cDNA by adding the following substances

2. Digest at 37°C for lh.

3. Incubate at 65°C for 20 min to heat inactivate the restriction enzyme.

4. Equal volume PCI extraction followed by ethanol precipitation (protocol A, step 2).

5. Resuspend the rinsed and air-dried precipitate in 20 ul LoTE.

6. Continue with step 4

Experimental program B

1. Remove the second strand of the chain reaction mixture from the test tube and wash it once with 50ul of wash solution, then remove the wash solution.

2. Wash the tube once with 50ul of 1x Restriction Buffer.

3. Remove the buffer and add:

4. Digest at 37C for lh.

5. Heat inactivate the restriction enzyme by placing at 65°C for 20 min.

6. Continue with step 5.

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