Exercise #8: PCR Cleanup

In this exercise, you will perform a PCR Cleanup to remove extra material from your PCR reaction so that you can move forward with a pure sample

Learning Objectives

Abilities

At the end of the activity, you will be able to:

• Purify your PCR sample

Skills

• Preparing reagents

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Instructions

Note: Do not use your control PCR tubes in this protocol as there is no DNA in them to purrify

1. Transfer the entire content of your PCR reaction mixture to a 1.5mL Eppendorf tube and add 5 volumes of Buffer B3

2. Transfer the above mixture solution to the EZ-10 column and let it stand at room temperature for two minutes. Centrifuge at 10,000rpm for 2 minutes.

3. Dispose of the flow through. Add 700uL of Wash Solution to the column and centrifuge at 10,000rpm for 2 minutes

5. Repeat step 4

6. Dispose of the flow through and spin again at 10,000 rpm for 1 minute to remove any residual Wash Solution

7. Transfer the EZ-10 column to a clean 1.5mL Eppendorf tube and add 30-50uL of Elution Buffer to the center of the column. Incubate at room temperature for 2 minutes. Centrifuge at 10,000 rpm for 2 minutes to elute the DNA.

8. Take the flow-through solution and pipette it back into the column.

9. Incubate at room temperature for 2 minutes and centrifuge at 10,000rpm for 2 minutes.

10. Repeat steps 8-9

11. Now discard the EZ-10 column - your DNA sample is in the liquid at the bottom of your Eppendorf.

Measuring DNA Concentration with a Spectrophotometer

1. Get new Eppendorf tubes and label them to correspond with your DNA samples

2. Add 3uL of your DNA sample into your new Eppendorf tubes

3. Add 297uL of distilled water to your Eppendorf's

4. Take your sample over to the spectrophotometer. First blank the machine by measuring a cuvette of 300uL of water and zeroing the machine. Then add your 300uL sample to the cuvette and record its A260 value