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Often the buffer conditions or reaction components in one cloning step are not compatible with the next, so a “PCR cleanup” is used to purify the DNA and remove these unwanted components. The DNA doesn’t necessarily need to be from a PCR reaction, and a PCR cleanup can be used to clean up restriction digests as well.
The PCR cleanup comes in a kit and we will use it to purify the DNA away from the buffer components and enzymes in our pET28b restriction digest before proceeding to the Gibson Assembly reaction.
- 1.5ml microcentrifuge tubes
- Zymo PCR Cleanup Kit
Centrifugation should be performed at approx. 10,000 g unless otherwise specified.
Note: Instructions provided are for the Zymo PCR cleanup kit. While all cleanup kits utilize similar protocols, there may be small but significant differences between manufacturers. If using another kit, please follow the instructions listed by the manufacturer for steps 1-6.
- In a 1.5mL microcentrifuge tube, add 2 volumes of Zymo DNA Binding Buffer to each volume of DNA sample (100uL buffer for a 50uL digest). Mix briefly by vortexing.
- Transfer the mixture to a Zymo-Spin Column in a Collection Tube.
- Centrifuge for 30 seconds. Discard the flow-through.
- Add 200uL of Zymo DNA Wash Buffer to the column. Centrifuge for 30 seconds. Discard the flow-through.
- Repeat step 4. After the second wash, discard the flow-through and spin 1 minute to remove any residual wash buffer.
- Add 10uL of water directly to the column matrix and incubate at room temperature for one minute. Transfer the column to a clean 1.5mL tube labeled “Digested pET28b vector” and centrifuge for 30 seconds to elute the DNA.
- Quantify your DNA using the Nanodrop. Record the concentration of DNA on the side of your tube.
Store DNA at 4°C until ready to use.