Join the joint experiment

Summary

The junction is constructed by allowing the two complementary primers to anneal, and the 5' ends of primers 1B and 2B should first be phosphorylated.

Modern Neuroscience Research Techniques

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

Operation method

Join the joint experiment

Materials and Instruments

10 mol L ATP PNK (9.3 U ul) 5 x T4 DNA Ligase Buffer LoTE

Move

1. Dilute primers IB and 2B to a concentration of 350 ng/V.

2. Add the following reagents for primer 5' end phosphorylation:

3. Incubate at 37℃ for 30 min.

4. Heat inactivate PNKo at 65°C for 10 min.

5. Add 36ul of primer IA to 80ul of phosphorylated primer 1B, and similarly add 36ul of primer 2A to phosphorylated 2B (final concentration 217ng/ul).

6. Anneal the primers: first heat to 95℃, hold for 2 min, then place at 65℃ for lOmin, 37℃ for 10 min, and then at room temperature for 20 min.

Note: A 200 ng junction can be taken for test ligation followed by 12% polyacrylamide gel electrophoresis. If phosphorylation is successful, most junctions (>70%) should ligate into dimers (±80bp).

Experimental protocol A

1. Attach junction 1 to one copy of NlaIII-enzymatically cleaved cDNA bound to Dynabeads and junction 2 to the other copy. Add the following on ice:

2. Heat to 50C, hold for 2 min, then leave at room temperature for 15 min to anneal the junction to the MaIII digested cDNA.

3. Add 2ul of T4 DNA ligase (5U/ul), then ligate the junctions at 16°C for 2 h.

4. After junctions are completed, rinse the beads 4 times with 2004 IX Binding and Rinse Buffer, and then rinse twice with 200ul of 1X Restriction Buffer.

5. After completing the last rinse step, immobilize the beads and remove the LoTE.

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