Lambda DNA Purification

This Protocol is for the Purification of Lambda DNA out of E. coli lysogens infected with lambda phage. This protocol spans multiple days and contains steps for lysogen confirmation, phage induction and finally DNA purification.

Contents

1. Before you start
2. Materials
3. Day one
4. Day two
5. Day three
6. Day four
   1. Setup
   2. Growth, induction and lysis
   3. Phage pellet and proteinase K
   4. Protein precipitation
   5. Clarification and pH adjustment
   6. Column purification and DNA precipitation
7. Gel Confirmation
8. Concentration Gel

1. Before you start

Tip

Some lysogens don’t have any antibiotic resistance. Use sterile technique and be careful not to contaminate your samples.

Danger

When making the buffers make sure you titrate well to the correct Ph, and re-titrate the day before your experiment. Lambda DNA will not bind to the purification column if it is not the correct pH.

Caution

You can freeze your lysogen pellet! It is highly recommended to make the days much more manageable.

2. Materials

List of materials needed:

• stable lysogen (Max has)
• LB-Agar plates
• LB (2X 50 ml overnight and 2X 500 ml grow up in 4L flasks)
• All buffers listed on the final page of the Qiagen lysogen DNA purification protocol
• Qiagen Tip-500 columns (Max has some for use)

3. Day one

1. Obtain lysogen and streak onto LB plates (no antibiotic).
2. Grow at 30°C overnight.

4. Day two

1. Streak 6 single colonies onto two 6-section plates.
2. Grow one plate at 30°C and one at 42‘overnight. * Streak on the 42°C plate first for a more definitive confirmation.

5. Day three

• Colonies that have grown on the 30°C sections and not on the 42°C sections are lysogens.

1. Inoculate 2X 50ml of LB with a single lysogen colony. Grow overnight at 30’
2. Autoclave 2X 4L flasks with 500 ml of LB. Put in 30’ shaker with inoculated overnight cultures for the next day use.

6. Day four

Tip

DNA is robust! during the pelleting and purification, you do not need to worry about keeping anything on ice unless specifically stated.

This is the long day, so try to get this started as early as you can.

6.1 Setup

1. Set up the following equipment:
   • 2 large water baths at 70°C
   • 1 small water bath at 55°C
   • 1 incubator at 45°C
   • 1 incubator at 30°C.
   • For each flask, put a large flask holder in both the top and bottom shaker incubator.
2. Ensure you have the following materials PER STRAIN:
   • 500ml LB in 4L flask (with antibiotic, if appropriate)
   • 1 large centrifuge bottle
   • 200ul Chloroform
   • Four (4) 15ml conical tubes
   • Seven (7) 50ml conical tubes
   • 250uL DNAse
   • 30uL RNAse
   • 40mL SM Buffer
   • 10mL Buffer L2
   • 3mL Buffer L3
   • 3mL Buffer L4
   • 30uL Proteinase K
   • 3mL Buffer L5
   • 1 Qiagen tip-500 column
   • 10mL Buffer QBT
   • pH strips
   • 20mL 1M MOPS pH 8.0
   • 30mL Buffer QC
   • 15mL Buffer QF
   • 10.5mL Isopropanol
   • 10mL 70% ethanol
   • 1mL Te

6.2 Growth, induction and lysis

1. Transfer 1ml of sterile LB into a cuvette to blank the UV-Vis.
2. Inoculate both 500ml grow up cultures with 10ml of overnight culture.
3. Shake the 4L flasks at 30°C for 2 to 2.5 hours until the OD is between 0.6 and 0.7.
4. Collect a 1ml aliquot from each liquid culture and set aside. Label it F1.
5. Put the flasks back in the 30°C incubator for just a few minutes to get them back to temperature.
6. Increase the temperature in both flasks rapidly to 42°C by swirling the flasks in a 70°C water bath:
   • Be extremely careful not to exceed 42°C!
   • Gently place a glass thermometer in each flask.
   • Remove the cap so that condensation does not form on the sides of the flask, blocking your view of the thermometer.
   • It may be easier to do one flask at a time.
7. After the temperature in both flasks is 42°C, place the flasks in a 45°C shaker and shake for an additional 15 minutes.
8. Increase the temperature in the previous incubator (which is now empty) to 37°C. By the time the flasks are done incubating in the 45°C incubator, this should be ready for the next step.
9. Transfer the flasks from the 45°C incubator to the 37°C incubator.
10. Shake cultures for an additional 2 hours.
11. Collect a 1ml aliquot from each liquid culture and set aside. Label it F2.
12. Transfer the cell broth to two large centrifugation bottles and centrifuge at 3000 G’s for 30 minutes.
13. Pour off the supernatant and suspend each pellet in 10ml of SM buffer.
14. Transfer the suspended cells into two separate 15ml falcon tubes.
15. If freezing the pellet move on to the next step. If you plan to do the purification all in one day skip to chloroform lysis step 23.
16. If storing the pellets, centrifuge the falcon tubes at 3000 G’s for 30 minutes.
17. Pour off the supernatent and flash freeze the pellets in liquid N2.
18. Store the pellets in the -80. Storage confirmed for maximum of ONE DAY.
19. When you are ready to re-start purification, remove the pellets and place in a room temperature water bath.
20. Top the pellets with 5ml of room temperature SM buffer. Let the pellets thaw for approximately 5 minutes.
21. Vortex the pellets to suspend. If chunks still persist shake the tubes.
22. Add 5ml of SM buffer to a total of 10ml.
23. Add 200ul of chloroform to each tube and vortex vigorously.
24. Incubate the tubes shaking on their side at 37°C for 30 min. After shaking the culture will be extremely thick. Start defrosting the DNAse and RNAse for the next step on ice.
25. Add *250uL (50ng/ul total) of DNAase and *30uL (30ng/ul) of RNAase to each tube. *Calculate your amount to add based off of initial concentration! Too much DNAse will shear lambda DNA.
26. Incubate the tubes shaking on their side at 37°C for 1 hour.
27. Centrifuge the tubes at 6000 G’s for 15 min.
28. Transfer the supernatant to a 50ml falcon tube.
29. Dilute the supernatant up to 40ml total volume with 30ml of SM buffer.
30. Collect a 500µl aliquot from each sample and set aside. Label it: F3
31. Add 10ml of ice cold buffer L2, and invert to mix.
32. Incubate the lysate on ice for 1 hour in the cold room.

6.3 Phage pellet and proteinase K

1. Place the conical tubes with the volume measurement labels facing outward (“laces out”) in the centrifuge. This is done so that the faint white phage head precipitate can be seen. If you have the white label part on the outward side, the phage heads won’t be visible.
2. Centrifuge at 10,000 G’s for 10 minutes. This will pellet the DNA containing phage heads. They will look like white smears and pellets on the sides of the tube.
3. Discard the supernatant.
4. Place the tubes upside down on a paper towel for 1 minute to drain any remaining liquid.
5. Suspend the small phage pellet in 3ml of buffer L3. Pipette up and down and around the walls of the tube to suspend all the phages on the tube walls.
6. Add 3mL of buffer L4.
7. Now add 30ul of proteinase K to a final concentration of 0.1 mg/ml.
8. Incubate the lysate at 55°C for 30 minutes and then incubate at 70°C for 10 minutes.

6.4 Protein precipitation

1. Add 3ml of buffer L5. This will instantly create a white precipitate.
2. Immediately swirl the solution GENTLY.
3. Gently transfer the lyaste to a 15ml falcon tube.
4. Centrifuge the lysate at 4°C for 30 minutes at 15000 G’s.

6.5 Clarification and pH adjustment

1. Transfer the supernatant to a new tube.
2. Repeat the previous centrifugation step for 10 minutes.
3. Collect a 90ul aliquot from each sample and set aside. Label it: F4
4. Equilibrate 2 Qiagen tip-500 columns with 10ml of buffer QBT(pH 7.0).
5. Test the pH of the lysate using pH strips. It will probably be 5.5. This is too low! The pH must be 7.0 for the DNA to bind to the column!
6. Titrate the pH to 7.0 by using 1M pH 8.0 MOPS buffer. You will use approximately one volume.
7. Confirm the pH of the lysate is 7.0 using pH strips. You are ready to begin the column separation of your phage DNA!

6.6 Column purification and DNA precipitation

Caution

The column steps can be very long, especially flow through and washing. If the prep was done correctly flow through will take overnight, and washing and elution will take most of the next day. This is OK because the columns are designed to never run dry. Fast columns mean little DNA.

1. Load each tube of lysate into its own column.
2. Wait for the lysate to completely flow through the column. If DNA is concentrated, this will take overnight.
3. Collect 90µl of the flow through from each sample and set aside. Label it: F5.
4. Wash each column with 30ml of buffer QC. This could take a few minutes to a few hours.
5. Warm the elution buffer QF to 65°C.
6. Collect 100ul of the wash eluent from each sample and set aside. Label it: F6.
7. Elute the DNA with 15ml of buffer QF (elution buffer, pH 8.5).
8. To speed up elution, seal the column/tube with parafilm and tape, cap the column with foil, and place in a dry incubator set to 70°C.
9. Collect 50ul of the final eluent from each sample and set aside. Label it: F7.
10. To precipitate the DNA, add 10.5 ml of isopropanol. If concentrated DNA is present in the elution, a hazy precipitate of stringy lambda DNA will visibly crash out.
11. Gently invert the tube several times to ball up the DNA.
12. Spool up the DNA on a bent pipette tip. (We use this way instead of centrifugation because this is more gentle, so better for microscope work.)
13. Wash DNA by dipping into 70% EtOH (x2).
14. Dry the DNA for 2-5 minutes until most of the residual ethanol is gone.
15. Suspend the pellet in Te buffer. Use just enough to dissolve the sample. For large pellets a little over 1ml Te is required.
16. Dissolve the DNA by heating for 1 hour in a 50°C dry block, or until you can’t see the DNA move around anymore.

7. Gel Confirmation

It should be clear if you have lambda during the isopropanol precipitation step. But it is also a very good idea to use the samples collected throughout the protocol to do a gel confirmation.

• For samples F1-F3 add 4% final of phenol-chloroform and spin down at 6000g for 5 min. Remove the aqueous layer and precipitate with 1.25 M final of sodium acetate and 1 volumes worth of isopropanol.
• For samples F4-F7, add 1 volume of isopropanol(IPA) to each sample.
• Centrifuge all samples at 15000g for 30 minutes and carefully decant the supernatant.
• Redissolve the DNA pellets in 10ul of TE.
• Run 5ul of each sample on a 0.8% agarose gel next to 20-200ng of NEB lambda.

8. Concentration Gel

If you successfully purified DNA, you will need to know what the concentration is. If your DNA is free of genomic DNA and mononucleotide contamination use the nanodrop. If contamination is present you will need to use a dilution serires with NEB Lambda used as a standard.