Revision as of 17:22, 20 October 2020

PROTOCOLS

PCR Cleanup

Reagents required:

• PCR clean up kit (E.N.Z.A)

• PCR reaction product

Procedure:

1. Transfer 30-50 μL of the PCR product to a new 1.5 mL microcentrifuge tube.

2. Add 4-5 times volume of the CP buffer.

3. Briefly vortex and centrifuge the samples.

4. Insert the Hibind DNA mini columns into the 2 mL collection tubes.

5. Add the samples from step 3 to the Hibind DNA mini column.

6. Centrifuge at 13000 g for 60 seconds at room temperature. Discard the filtrate and re use the collection tubes.

7. Add 700 μL of DNA wash buffer diluted with ethanol to the tubes.

8. Centrifuge at maximum speed for 60 seconds. Discard the filtrate and re use the collection tubes.

9. Repeat step 7 for a second DNA wash buffer step.

10. Centrifuge the empty Hibind DNA columns at maximum speed for 2 minutes to dry the column. This step is critical to remove the trace ethanol.

11. Transfer the Hibind DNA column to a new 1.5 mL microcentrifuge tube.

12. Add 50 μL of the elution buffer directly into the Hibind DNA column.

13. Keep at room temperature for 2 minutes and centrifuge at maximum speed for 60 seconds.

14. Store the DNA at -20℃.

Plasmid DNA Extraction

Plasmid DNA Extraction Reagents required: • E.N.Z.A Plasmid DNA mini prep kit • O/N Culture Procedure: 1. Vortex the liquid cultures and use 1-5 mL for the plasmid extraction. 2. Centrifuge at 10000 g for 1 min at room temperature. Discard the pellet. 3. Add 250 μL of solution I, vortex the samples and transfer the suspension to a new microcentrifuge tube. 4. Add 250 μL of solution II, invert the samples and wait for 2-3 minutes. 5. Add 350 μL Solution III and invert the tube several times. 6. Centrifuge the samples at maximum speed for 10 minutes. 7. Place the Hibind DNA columns onto the 2 mL collection tubes. 8. Transfer the supernatant from step 6 into the Hibind DNA columns. 9. Centrifuge at maximum speed for 1 minute and discarded the filtrate. 10. Add 500 μL of HBC buffer and centrifuge it at maximum speed for 1 minute and discard the supernatant. 11. Add 700 μL DNA Wash Buffer and centrifuge it at maximum speed for 30 seconds and discard the supernatant. 12. Repeat step 11 for a second DNA wash step. 13. Centrifuge the empty Hibind DNA column for 2 min at maximum speed to dry the column.This will remove trace amounts of ethanol. 14. Transfer the Hibind DNA column into a new microcentrifuge tube. 15. Add 50 μL of Elution Buffer, let it sit at room temperature for 2 minutes and centrifuge at maximum speed for 1 minute. 16. Store DNA at -20℃.

Reagents required:

E.N.Z.A Plasmid DNA mini prep kit
O/N Culture

Procedure:

Vortex the liquid cultures and use 1-5 mL for the plasmid extraction.
Centrifuge at 10000 g for 1 min at room temperature. Discard the pellet.
Add 250 μL of solution I, vortex the samples and transfer the suspension to a new microcentrifuge tube.
Add 250 μL of solution II, invert the samples and wait for 2-3 minutes.
Add 350 μL Solution III and invert the tube several times.
Centrifuge the samples at maximum speed for 10 minutes.
Place the Hibind DNA columns onto the 2 mL collection tubes.
Transfer the supernatant from step 6 into the Hibind DNA columns.
Centrifuge at maximum speed for 1 minute and discarded the filtrate.
Add 500 μL of HBC buffer and centrifuge it at maximum speed for 1 minute and discard the supernatant.
Add 700 μL DNA Wash Buffer and centrifuge it at maximum speed for 30 seconds and discard the supernatant.
Repeat step 11 for a second DNA wash step.
Centrifuge the empty Hibind DNA column for 2 min at maximum speed to dry the column.This will remove trace amounts of ethanol.
Transfer the Hibind DNA column into a new microcentrifuge tube.
Add 50 μL of Elution Buffer, let it sit at room temperature for 2 minutes and centrifuge at maximum speed for 1 minute.
Store DNA at -20℃.

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