Combined with magnetic bead experiments

Summary

This step is only part of Experiment A in the cDNA synthesis for serial analysis of gene expression. In Experiment B, the cDNA is already bound to the streptavidin-antibiotin-coated PCR tubes and therefore does not need to be bound with magnetic beads.

Modern Neuroscience Research Techniques

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

Operation method

Combined with magnetic bead experiments

Materials and Instruments

Magnetic Beads
Dynabead M-280 Streptavidin Antibiotics
Magnetic Devices

Move

Experimental program A

1. Shake for lmin to reconstitute the Dynabead M-280 Streptavidin anti-biotin into a suspension.
2. Transfer 2X of 100 ul Dynabeads into U-unit 1.5 ml Eppendorf tubes.
3. Fix the beads with a magnetic device and remove the supernatant.
4. Resuspend the beads 3 times with 200 ul of IX Binding and Rinse Buffer, immobilize the beads, and remove the rinse solution.
5. Divide the NlaIII digested cDNA into two 10ul portions, add 90ul of redistilled water and 100ul of 2X Binding and Flushing Buffer to each portion; add one portion of the rinsed beads to the mixture.
6. After mixing, incubate at room temperature for 30 min (mixing every IOmin).
7. The beads were immobilized with a magnetic device and the supernatant was discarded.
8. Rinse the immobilized beads 3 times with 200ul of IX Binding and Rinse Buffer, then rinse once with 200ul of LoTE.
9. After the last rinse, immobilize the beads and remove the LoTE.
10. Proceed to step 5

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