EZ96?M13IsolationSpinProtocol

實驗概要

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.

主要試劑

1. Sterile deionized water (or TE buffer)

2. Absolute (96%-100%) ethanol

主要設備

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

實驗步驟

1. Grow the M13  infected bacteria in a 2.2 mL 96-well culture plate (not supplied). Spin  down bacterial cells by centrifugation at 5000 rpm for 15 minutes at  room temperature.

2. Transfer 1mL 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.

3. 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  10-15 minutes.

4. Transfer 1 mL of the cleared supernatant from step 3 into each well of the E-Z^? 96 DNA Plate. Place E-Z^?  96 DNA Plate onto 2 ml collection Plate with tape or film. Place  plate/2mL collection tube together in centrifuge’s swing-bucket rotor  with adapter for deep well plate. Centrifuge at 3,000 x g for 5 minutes.  Discard the liquid.

5. Add 1 mL of Buffer MPX to each well of the E-Z 96^? DNA  Plate. Immediately Centrifuge at 3000 x g for 5 minutes. Discard the  flow-through and reuse the 2 mL collection tubes for next step.

6. Add another 1 mL of buffer MPX to each well of the E-Z 96^?  DNA Plate. Incubate for 2 minutes at room temperature. Centrifuge at  3000 x g for 5 minutes. Discard theflow-through and reuse the 2 mL  collection tube for next step.

7. Add 1 mL of SPW Wash Buffer into each well of the E-Z 96^? DNA Plate. Centrifuge at 3000 x g for 15 minutes. Discard the flow-through and reuse the 2 mL collection tube for next step.

8. (Optional) Place E-Z 96^? DNA Plate into a vacuum oven  or incubator which was preset to 70°C for 10 minutes. This step will  ensure that the DNA plate is completely dried before DNA elution.

9. Carefully place the E-Z 96^? DNA plate on top of the new  Racked Microtubes (supplied). Add 75-150ul Water or Elution Buffer  (10mM Tris, pH8.5 ) to each well of the E-Z 96^? DNA Plate. Let stand for 2 minutes.

10. Centrifuge at 3000 x g for 5 minutes to elute DNA and seal the  tube with caps. This represents approximately 75-80% of bound DNA. An  optional second elution will yield any residual DNA, though at a lower  concentration.