Lambda QUICK!^® SPIN Kit Protocol
Revision No. 2055-999-5G02W

The Lambda QUICK! SPIN^TM Kit is designed for rapid isolation and purification of lambda phage DNA from infected/lysed bacteria cultured on agar plates. Once cell lysis is complete, phage is harvested and purified. The phage particles are then lysed and phage DNA is isolated and purified with Lambda QUICK! SPIN^TM Matrix. The procedure saves time and reagent cost; no cesium chloride centrifugation is required. The protocol can be completed in less than two hours, yielding an average of 20 µg Lambda DNA per lysed plate. The Lambda QUICK! SPIN^TM Kit contains all reagents (except for 100% ethanol, isopropanol, and TE) required for this procedure and is available in three sizes for 10, 25 and 100 phage DNA purifications. The reagents and materials provided with this product are for research purposes only.

1. Grow overnight culture of lambda-sensitive E.coli strain in broth containing 10 mM MgSO₄ and 0.2% maltose [i.e. NZCYM Broth, BIO 101, Cat. No. 3004-011 (capsules) or 3004-012 (powder); 20% Sterile Maltose, BIO 101, Cat. No. 3068-034 (solution) or 4006-012 (powder)].


2. Pellet cells by centrifugation at 4,000x g for 5 minutes. Discard supernatant and resuspend cells in 1/4 volume of 10 mM MgSO₄.


3. Prepare phage dilutions as follows: Add 10 µl of phage stock to 10 ml SM (10-3 dilution). Add 10 µl of the 10-3 dilution to 10 ml SM (10-6 dilution). Use 10-6 dilution to titrate the phage.


4. Prepare two tubes containing 0.2 ml prepared E.coli cells (from step 1 above). Using the 10-6 phage dilution, add 10 µl to tube 1 (10-8 dilution) and 100 µl to tube 2 (10-7 dilution). Mix and incubate at 37°C for 10 minutes.


5. To each tube, add 3.5 ml of melted (50-55°C) top agar, mix by inverting once, and pour onto prewarmed (37°C) LB or NZYCM Agar plate. Gently tip plate back and forth to spread top agar evenly. Let top agar harden; then invert plates and incubate at 37°C overnight.


6. Count number of plaques and calculate titer of the phage stock.

Example 1: 300 plaques on 10^-8 dilution plate => 3x10¹⁰ pfu/ml phage stock.
Example 2: 500 plaques on 10^-7 dilution plate => 5x10⁹ pfu/ml phage stock.

1. Grow a fresh overnight culture of lambda-sensitive E.coli at 37°C in broth (see A1, above).


2. Pour plates (25 ml per 85 mm plate, 50 ml per 150 ml plate) with Lambda QUICK Bottom Agar (supplied) as per instructions on label. For every 85mm plate, infect 100 µl cells with 105 pfu in a sterile Falcon 2059 or similar tube and incubate at 37°C for 10 minutes. Prepare one tube for each plate.


3. To each tube, add 3.5 ml top agarose (as supplied) at 45-50°C; mix; pour onto a 37°C 85mm plate containing bottom agar and allow to solidify. Incubate at 37°C for 8-10 hours or until cell lawn is lysed nearly confluently. (Instructions in steps 2 and 3 are for one 85mm plate. If 150 mm plates are used, double all values in steps 2 and 3. Only two 85mm or one 150mm plate will be necessary per typical prep.)

1. Scrape top agar from plate(s) into a centrifuge tube and spin at 10,000 xg for 10 minutes. Pour liquid through Sieve Cloth into clean 50 ml tube and save aside.


2. Add 8 ml of Lambda QUICK! Suspension Buffer to agarose pellet. Vortex for 1-2 minutes. Spin at 10,000x g for 10 minutes. Remove supernatant and combine with supernatant saved aside in step 4 above. Discard agarose pellet.


3. Precipitate phage by adding an equal volume of isopropanol (user-supplied); mix thoroughly; spin at 10,000x g for 10 minutes. Decant supernatant; re-spin briefly to collect residual liquid in the bottom of the tube, and remove.


4. Resuspend pellet with 1.2 ml TE buffer (10 mM Tris, pH 7.5; 1 mM EDTA, user-supplied) and transfer to microcentrifuge tube. Spin at maximum speed for 2 minutes and transfer supernatant to new tube. Discard pellet.


5. Add 5 µl Lambda QUICK! Nuclease Mixx to resuspended phage. Incubate at 37°C for 15 minutes.


6. Add 1/3x volume of Lambda QUICK! Phage Drop Buffer (i.e. to 1.2 ml suspended phage, add 0.4 ml); mix, and incubate at 37°C for 20 minutes to precipitate phage.


7. Centrifuge at 10,000 xg for 5 minutes and discard supernatant. Re-spin briefly and remove traces of Lambda QUICK! Phage Drop Buffer.


8. Resuspend phage pellet in 150 µl TE buffer. Transfer resuspended phage to Lambda QUICK! SPIN^TM Filter and add 200 µl Lambda QUICK! SPIN^TM Matrix; mix thoroughly with a wide bore pipet tip, and incubate at 70°C for 10 minutes to lyse phage and allow DNA to bind to Matrix.


9. Spin for 1 minute in microcentrifuge at maximum speed and discard supernatant in catch tube.


10. Wash Lambda DNA/Lambda QUICK! SPIN^TM Matrix pellet by adding 300 ml Lambda QUICK! SPIN^TM Wash (remember to add an equal volume 100% ethanol to the Lambda QUICK! SPIN^TM Wash Concentrate before initial use) and spin for 2 minutes in microcentrifuge (or until all liquid is in the catch tube). Discard liquid in catch tube.


11. Repeat wash as described in step 13; discard liquid in catch tube. Re-spin for 1 minute to "dry" pellet and transfer Lambda QUICK! SPIN^TM Filter to a clean Lambda QUICK! Catch Tube.


12. To elute Lambda DNA, add 100 µl Lambda QUICK! Elution Solution and resuspend pellet gently by stirring with pipet tip; finger flicking of tube is also effective. To prevent shearing of the lambda DNA, do not pipet the suspension in and out through a pipet tip without first snipping off the tip. Be VERY careful with pipet tips not to pierce SPIN Filter membrane. Incubate at 70°C for 10 minutes. Spin for 2 min. in microcentrifuge (or until all liquid is in the catch tube). Repeat elution at least once or twice to maximize yield.

OD Readings
If DNA is resuspended in H₂O, add 5 µl TE per 500 µl to accurately measure the OD260/280.

Sequencing of Lambda DNA
Wash the Lambda DNA/Lambda QUICK! SPIN^TM Matrix pellet with 300 µl of a 1:1 solution of acetone:ethanol prior to the wash step in 10 to optimize the ability to sequence the Lambda DNA.

Matrix Overload Use one 85 mm plate per prep. If the Lambda QUICK! SPIN^TM Matrix appears viscous during the elution step and the flow-rate through the spin column is slow, the Lambda QUICK! SPIN^TM Matrix is probably overloaded and less phage (fewer plates) should be used. Nevertheless, the quality of the Lambda DNA from such a prep is quite adequate for most further modifications, restriction digestion, subcloning, etc.