EZ96®M13IsolationVacuumManifoldProtocol

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. Prepare 4 mL of an infected M13 culture, and incubate with vigorous shaking for 6-7 hours ours at 37°C.

2. Pellet bacteria by centrifugation at 5,000 rpm for 15 min at room temperature.

3. Transfer 1.4 mL of the supernatant obtained containing the M13  bacteriophage, into a fresh reaction tube. Be careful not to disturb the  bacterial pellet during the transfer. If the supernatant is not clear,  repeat the centrifugation step.

4. Add 280 ul of MPG Buffer to the M13 supernatant and mix by vortexing. Then, incubate at room temperature for 10-15 minutes.

5. Place OBI’s HiBind^® M13 Minicolumn into a 2 mL collection tube and add 700 ul of the M13 sample to the column.

6. Centrifuge at 10, 000 rpm for 30 seconds and discard the flow-through collected in the collection tube.

7. Repeat steps 5 and 6 until all of the sample has been passed through the HiBind^® M13 Minicolumn.

8. Lyse and bind the DNA onto the membrane by adding 700 ul of MPX buffer to the HiBind^® M13 Minicolumn.

9. Immediately centrifuge for 30 seconds at 10,000 rpm. Discard the flow-through and reuse the collection tube.

10. Apply another 700 ul of MPX buffer to the HiBind^® M13  Mini-column and incubate at room temperature for 1 minute. Centrifuge  for 30 seconds at 10,000 rpm. Discard the flow-through from the  collection tube and reuse the collection tube.

11. Add 700 ul of SPW Wash Buffer diluted with absolute ethanol to the HiBind^® M13 Minicolumn, and centrifuge it for 30 seconds at 10,000 rpm.

Note: SPW Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for instructions.

12. Discard the flow-though and wash the column with another 700 ul of SPW Wash Buffer by repeating step 11.

13. Discard the flow-through in the collection tube, and reuse the  collection tube by placing the column into the collection tube and  centrifuging at maximal speed ($13,000 rpm ) for 1 minute in order to  dry the column.

14. Place the dried HiBind^® M13 Minicolumn into a clean  1.5 mL microcentrifuge tube, and add 50-100 ul of Elution Buffer  (preheated at 60°C, 10mM Tris, pH8.5 ) to the center of the membrane.  Incubate at room temperature for 10 minutes and centrifuge for 1 minute  at maximal speed ($13,000 rpm). 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, make sure the pH of the  water or TE is between 7.5 -8.0.

15. Yield and quality of DNA: Determine the absorbance of an  appropriate dilution (20-to 50-fold) of the sample at 260 nm and then at  280 nm.