Exercise 4: Mini-Preps

In this exercise, you will learn how to perform a mini-prep in order to extract and purify DNA from cell cultures, and how to use a Spectrophotometer in order to measure DNA concentration.

Learning Objectives

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

Abilities

• Perform a glycerol stock for long term DNA storage

• Perform a mini-prep in order to extract and purify DNA from cell cultures

• Use a Spectrophotometer in order to measure said DNA concentration

Skills

• Following a given protocol

• Using a Spectrophotometer

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Materials:

• Overnight Liquid Culture

• 1.5mL centrifuge tubes

• Table-top centrifuge

• Vortexer

• EZ-10 Columns

• Mini-prep Solutions

• Spectrophotometer or Nanodrop to measure DNA concentration

Instructions:

1. Retrieve your overnight culture vials. The liquid in the sample vials should be colored while your negative control should still be clear.

Overnight cultures 18 hours later. Negative control on the far left is clear as no bacterial growth was seen. The middle two represent sample in 5mL of media resulting in a concentrated bacterial population. The right-most tube is bacterial growth in 10mL of media resulting in a less concentrated color

2. Retrieve one 1.5mL Eppendorf tube for each overnight culture vials you prepare. Label each centrifuge tube to correspond with one of your cultures. Eppendorf's 1 and 2 will be for your Red-CHL overnight cultures and Eppendorf 3 will be for your Blue-Kan overnight culture

3. Pour ~1mL of your overnight culture into the corresponding Eppendorf tube. This doesn't have to be exactly 1mL - just want to pour some of the culture into the tube without overflowing it. If you pour too much to the point where the lid won't close you can pore some of your culture back into your vial.

4. Centrifuge your samples at 12,000rpm for 2minutes. After this drain the liquid into the chemical waste container

5. Repeat steps 3-5 by pouring more of your culture into the same Eppendorf tube until all your overnight culture has been centrifuged

6. You will end up with a pinkish (or purplish) pellet at the bottom of your Eppendorf tube

Note: When using a centrifuge ensure that your samples are placed in a balanced way. If you have two samples, place on opposite sides of the machine. If you have an odd number of samples fill another Eppendorf tube with water and use as a counterbalance. Please ask for your instructor to double check your set-up if uncertain.

Ensure that your centrifuge is always balanced

7. Add 200μL of Solution I to each Eppendorf tube. Mix using the table-top vortex until the solid pellet at the bottom of the tube has been completely dissolved. To see if the pellet has been dissolved, flip the tube upside down and see if anything remains stuck at the bottom.

8. Add 400μL of Solution II to the mixture and mix gently by inverting the tube 4-6 times and keep at room temperature for 1 minute. Do not Vortex. The solution will now turn yellow throughout

10. Add 700uL of Solution III and mix gently via inverting and then leave at room temperature for 1 minute. This will create a clear solution at the bottom with a opaque white component on top with some yellow still in it. Invert until the yellow component is no longer visible

11. Centrifuge at 12,000rpm for 5 minutes. You will now have a white solid on the sides of your Eppendorf. This is excess waste that you do not want to carry forward. Be very careful over the next step to not disturb the pellet.

12. Set up your station with a number of EZ-10 column corresponding to the number of Eppendorf's you just centrifuged. Number the EZ-10 columns to correspond with your Eppendorf's.

13. Transfer half the supernatant (the liquid after a centrifugation) (About 650uL) into the corresponding EZ-10 column.

14. Centrifuge at 10,000rpm for 2 minutes and discard the flow-through.

15. Add the remaining supernatant to the EZ-10 column. Be very careful to not disturb any of the material on the walls of the tube. Its better to leave some liquid behind then disturb the waste and transfer some of it into your EZ-10 column

16. Centrifuge at 10,000rpm for 2 minutes and discard the flow-through.

17. Add 700uL of Wash Solution to the EZ-10 column and let rest for 2min before centrifuging at 10,000rpm for 2 minutes

18. Discard the flow-through and repeat step 17

19. Discard the flow-through and centrifuge again at 10,000rpm for 1 minute to remove any residual Wash Solution

20. Discard the flow-through

21. Transfer the EZ-10 column to a new clean 1.5mL Eppendorf tube. Number the lids of the Eppendorf columns to correspond with the EZ-10 column in it. You can dispose of the collection tube.

22. Add 50uL of Elution Buffer into the center of the column and incubate at room temperature for 2 minutes.

23. Centrifuge at 10,000rpm for 2 minutes

24. Take the flow-through solution and transfer it back into the column.

25. Incubate at room temperature for 2 minutes

26. Centrifuge at 10,000rpm for 2 minutes.

27. Repeat steps 24-26

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

Solution	Components	Purpose
I	Glucose EDTA Tris-HCl RNAse	Removes the bacterial cell wall and converts bacteria into spheroplasts RNAse also serves to denature any RNA present in the cell.
II	NaOH SDS	​Lyses the bacteria and denatures plasmid and chromosomal DNA SDS also denatures proteins
III	Potassium acetate Acetic acid	Acid neutralizes the base from solution II, allowing the plasmid DNA to renature. Potassium acetate causes cell debris and chromosomal DNA to precipitate out of the solution
Wash Buffer	​Ethanol Tris-HCl	​Washes out any additional cell debris from the solution, while the DNA remains trapped in the silica column.
Elution Buffer		Releases DNA from the silica column

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