clonal tandem assay

Summary

It is a complementary experiment to experimental protocols A and B of the differential display technique (DD) experiment and the gene expression series analysis experiment.

Modern Neuroscience Research Techniques

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

Operation method

clonal tandem assay

Materials and Instruments

Solutions & Buffers LoTE cDNA Label Ligase
Kits PCR Instruments

Move

I. Tandem ligation at the I site of pZero

1. SphI digest vector, ligated to size-recovered tandem fragments; we recommend cloning the tandem at the Sph1 site of pZero, and we refer to the Zero Background Cloning Kit manual for details on cloning in this vector:

2. Incubate at 16°C overnight.

3. Amplify volume to 200ul with LoTE.

4. Equal volumes of PCI were extracted (Experimental protocol A, step 2).

5. The following substances were added to precipitate the aqueous phase:

6. centrifuge in a microcentrifuge at 13,000 r/min for 15 min at 4°C.

7. Rinse the sediment 4 times (!!!) , then look dry.

8. Resuspend in 4ul of LoTE.

9. Transform ELECTROMAXDHlOB cells with 1ul of ligation mixture.

II. Electroporation

Any transformation method can be applied, and we recommend the electroporation method due to its high transformation efficiency ( ^>1010 transformants/ug plasmid) in binding to receptorized ELECrR0MAXDHlOB cells.

1. Melt 100ul of ELECTROMAX DH10B cells on ice.

2. Simultaneously cool the desired number of electroporation minicups on ice.

3. Immediately after melting, perform the next step on ELECTROMAXDH10B cells by dividing them into 35ul each (on ice).

4. Add ImI's ligation product to the 35ul-portion of cells and tap the tube to mix.

5. Transfer to a cooled beaker, move the cells between the electrodes of the beaker, and store on ice.

6. Electroporate the cells on the GenePulserD with the following settings:

7. Immediately after applying this pulse, add 965ulSOC media.

8. Incubate at 37°C for Ih.

9. Spread LB plates containing Zeocin antibiotic with 50ul or IOOul.

Note: Details of the plates and SOC media are described in the ZeroBackground Cloning Kit manual.

10. Incubate the lined plates overnight at 37°C in a thermostat.

11. The next day these plates should contain several hundred clones.

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