实验概要

The  E.Z.N.A.™ family of products is an innovative system that radically  simplifies the extraction and purification of nucleic acids from a  variety of sources. The key to this system is Omega Bio-Tek, Inc.’s  (OBI) new HiBind^® matrix that specifically, but reversibly,  binds DNA or RNA under certain optimal conditions, while allowing  proteins and other contaminants to be removed. The nucleic acids bound  to OBI’s HiBind^® Matrix are easily eluted with deionized water or a low salt buffer, and then suitable for any downstream application.

OBI’s  E.Z.N.A.™ M13 Kits are designed to purify up to 10ug of single-stranded  DNA from up to 3mL of phage supernatant. Yields of single-stranded DNA  obtained using E.Z.N.A.™ M13 Kits are around 3-10 ug and reproducible  when the isolations are performed from the same culture.

The E.Z.N.A.™ M13 procedure first calls for the infected bacterial  culture to be centrifuged to pellet the bacterial cell, and then MPG  buffer is added to the supernatent to precipitate the phage particles.  Next, the samples are loaded on to HiBind^® columns or on to E-Z^® 96 plates. The specially designed HiBind^® matrix will retain intact phage particles. These phage particles will then be lysed and bound to the HiBind^®  membrane after the addition of MPX Buffer. Finally, contaminants such  as protein are efficiently washed away with DNA Wash buffer, and pure  ssDNA is eluted with TE or water.

主要试剂

1. Sterile deionized water (or TE buffer)

2. Absolute (96%-100%) ethanol

主要设备

1. Microcentrifuge capable of at least 10,000 x g

2. Sterile 15 mL centrifuge tubes

3. Sterile 1.7 mL centrifuge tubes

4. Water bath preheated at 60°C

实验步骤

1. Grow the M13 infected bacteria in a 2.2 mL 96-well culture plate (not supplied).

2. Spin down bacterial cells by centrifugation at 5000 rpm for 15 minutes at room temperature.

3. Transfer 1-2 mL of the supernatant into a 2 mL Collection Plate  (supplied). Be careful not to disturb the bacterial pellet during the  transfer. If the supernatant is not clear, repeat the centrifugation  step.

4. Add 1/5 volume of MPG Buffer (200μl MPG per 1 mL culture) to the  M13 supernatant and mix by vortexing. Incubate at room temperature for  15 minutes.

5. Assemble the vacuum manifold by following the manufacturer’s  instructions. If using Vac-03 vacuum manifold, place the waste  collection tray inside the manifold and place the E-Z^® 96 DNA Plate on top part of the manifold.

6. Transfer 1 mL of the cleared supernatant from step 4 into each well of the E-Z^®  96 DNA Plate. Switch on the vacuum for 2 minutes to draw the sample  through. Note: Load 1mL of the sample at a time. If the volume of the  sample is more than 1mL, ventilate the vacuum manifold and load another  1mL of the sample into each well of the E-Z^® 96 DNA Plate.

7. Add 1 mL of buffer MPX to each well of the E-Z 96^® DNA Plate. Immediately apply the vacuum to draw all of the samples through the membrane.

8. Stop the vacuum and add another 1 mL of buffer MPX to each well of the E-Z 96^® DNA Plate. Incubate for 2 minutes at room temperature.

9. Next, apply the vacuum until all of the liquid passes through the membrane.

10. Add 1 mL of SPW Wash Buffer into each well of the E-Z 96^® DNA Plate and switch on the vacuum until all of the liquid has passed through the membran.

11. Remove the E-Z 96^® DNA Plate from the vacuum manifold  and remove any traces of liquid by tapping the E-Z® 96 DNA Plate firmLy  on a stack of paper towels. Visually inspect the walls of the wells and  make sure that all droplets are removed by tapping.

12. Place the E-Z 96 DNA Plate back on the manifold and apply the  vacuum for another 5 minutes. Repeat the tapping step from step 11 to  completely remove any remaining liquid.

13. Optional: Place the E-Z^® 96 DNA Plate into a vacuum oven preset at 60°C and incubate for 10 minutes to completely dry the plate.

14. Re-assemble the vacuum manifold by now replacing the waste  collection tray with a 1.2 mL rack of microtubes (supplied). Place the  E-Z^® 96 DNA Plate on the top part of the manifold.

15. Add 75-150ul of preheated (60°C) Elution Buffer (10mM Tris-HCl,  pH 8.5) into the center of the membrane to each well of the E-Z^®  96 DNA Plate. Incubate for 10 minutes. Apply the vacuum for 5 minutes  to elute DNA. This represents approximately 75-80% of bound DNA. An  optional second elution will yield any residual DNA, though at a lower  concentration. The pH of the elution solution can also significantly  affect the elution efficiency , therefore make sure that the pH of the  water or TE is between 7.5-8.0.