E.Z.N.A.?FastfilterPlasmidMegaProtocol

實驗概要

The  E.Z.N.A.? family of products is an innovative system that radically  simplifies extraction and purification of nucleic acids from a variety  of sources. Key to the system is the Omega Bio-tek’s proprietary HiBind^?  matrix that avidly, but reversibly, binds DNA or RNA under certain  optimal conditions allowing proteins and other contaminants to be  removed. Nucleic acids are easily eluted with deionized water or low  salt buffer.

The E.Z.N.A.?  Fastfilter Plasmid Mega Kit combines time-tested consistency of  alkaline-SDS lysis of bacterial cells with Omega Bio-tek’s innovative  high efficiency DNA binding technology to recovery large scale high  quality plasmid DNA. This new methods facilitate the binding, washing,  and elution steps thus enabling multiple samples to be simultaneously  processed. This kit uses a Syringe-Format System is designed to replace  the centrifugation step following alkaline lysis of bacterial cells, the  Lysate Clearance Filter Syringes completely remove SDS precipitates and  clear bacterial lysates in a fraction of the time required for  centrifugation. Yields vary according to plasmid copy number, E.coli  strain, and conditions of growth, but 500 mL of overnight culture in LB  medium typically produces 2.5 mg high-copy plasmid DNA. Up to 1 liter  overnight culture may be processed when working with low-copy number  plasmids. The product is suitable for automated fluorescent DNA  sequencing, restriction endonuclease digestion, transfection of  mammalian cells, and other manipulations.

This Protocol is designed to isolate 2.5 mg of high Copy-Number  plasmids or 200 -500μg of low Copy-Number Plasmids from 500 mL overnight  cultures using E.Z.N.A.? Plasmid Mega Kit.

主要試劑

Absolute ethanol (96-100%)

主要設備

1. Centrifuge with swinging bucket rotor capable of 5,000 x g with adapter for 50 ml centrifuge tube

2. Vacuum pump capable of generate -200 to -600 mbar

3. Vacuum manifold with standard leur connector

4. 50 ml Centrifuge tube

5. 125 or 250 mL Bottle/Centrifuge

6. Centrifugation tube (i.e Nelgene 3120)

7. Vacuum Manifold (Cat No. Vac-08)

8. Ice

實驗步驟

Growth of bacterial culture:

1. Culture volume: Inoculate 500 mL LB/ampicillin (50 ug/mL) medium placed in a 1-5 liter culture flask with E.coli carrying  desired plasmid and grow at 37°C with agitation for 12-16 h. For best  results use overnight culture as the inoculum. It is strongly  recommended that an endA negative strain of E.coli be used for routine plasmid isolation. Examples of such strains include DH5α^? and JM109^?.

Note: Optimal growth conditions of bacteria is vital of obtaining  maximal plasmid DNA yields. The best conditions are achieved by picking a  single isolated colony from a freshly transformed or freshly plate to  inoculate a 2-5mL starter culture containing the appropriate antibiotic.  Incubate for ~8 hours at 37°C with vigorous shaking (~300rpm). Then  used to inoculate appropriate volume of Pre-warmed liquid growth medium  with antibiotic. Grow at 37°C for 12-16 hr with vigorous  shaking(~300rpm).Using a flask or vessel with a volume of a least 3-4  times the volume of the culture and dilute the starter culture 1/500 to  1/1000 into growth medium. 600 Following overnight bacterial growth, an  OD of 1.5~2.0 indicates a well-grown culture. For the 600 best result  determination of OD for each culture is recommended. it is important to  dilute the bacterial culture (10 to 20 fold) to enable photometric  measurement in the linear range between 600 600. 0.1 and 0.5 OD . We  recommend a bacterial density of between 2.0 and 3.0 at OD When using  nutrient-rich media, care should be taken ensure that the cell density  does not exceed 600 an OD of 3.0.

If using a frozen glycerol stock as inoculum, streak it onto an agar  plate containing the appropriate antibiotic for single colony isolation.  Then picking a single colony and inoculate the 2-5mL starter culture as  described above.

Lyse bacterial cells with alkaline-SDS Solution:

2. Pellet up to bacteria in appropriate vessels by centrifugation at 5,000 × g for 10 min at room temperature.

3. Decant or aspirate medium and discard. To ensure that all traces  of the medium are removed, use a clean paper towel to blot excess liquid  from the wall of the vessel.

4. Add 20 mL Solution I/RNase A. Resuspend cells completely by vortexing or pipetting up and down.

Note: Complete resuspension of cell pellet is vital for obtaining good yield.

5. Add 20 mL Solution II, gently mix by inverting and rotating tube 7  times to obtain a cleared lysate. Incubate 3 minutes at room  temperature.

Note: Avoid vigorous mixing as this will shear chromosomal DNA and  lower plasmid purity. Prolonged incubation may lead to nicking of  plasmid DNA. (Store Solution II tightly capped when not in use.)

6. Add 20 mL Ice-cold Neutralization Buffer, cover, and gently mix by  inverting tube several times until a flocculent white precipitate  forms.

Note: The mix must be mixed throughly. If the mixture appears still  viscous, brownish and conglobated, more mixing is required to completely  neutralize the solution. Complete neutralization of the solution is  vital of obtaining good yields. Increasing centrifugation speed is  helpful to completely remove the precipitated bacterial cell material.  After centrifugation, a tightly packed cell debris pellet indicates  efficient lysis.

7. Clear the lysate using Fastfilter Lysate Clearance Syringe:  Immediately pour the lysate into the barrel of the Lysate Clearance  Filter Syringe. Allow the cell lysate to sit for 2 minutes. The white  precipitate should float to the top.

8. Hold the Lysate Clearance filter syringe barrel over the 125 or  250 m bottle/l tube and gently insert the plunger to expel the cleared  lysate to the tube. Some of the lysate may remain in the flocculent  precipitate, do not force this residual lysate through the filter.

Purify the plasmid with HiBind^? Mega Column:

9. Measure the volume of the supernatant, add 1/3 volume of the PFC Binding Buffer. Mix throughly by vortexing.

10. Insert a HiBind DNA Mega column to the vacuum manifold.

11. Pour the cell lysate from step 9 into the HiBind^? DNA  Mega column and turn on the vacuum source to draw all the liquid through  the column. Keep pouring the lysate until all the cell lysate pass  through the column. Turn off the vacuum source.

12. Two wash the DNA, add 20 ml PFW Wash Buffer to the HiBind DNA  Mega column and aply the vacuum to draw all the liquid through the  column.

13. To wash the DNA, add 20 ml DNA Wash Buffer to the HiBind^?  DNA Mega column and apply the vacuum to draw all the liquid through the  column. Keep adding additional 20 ml DNA Wash Buffer until all the  liquid pass through the column.

14. Transfer the HiBind^? DNA Mega column into a 50 ml centrifuge tube (supplied). Centrifuge at 5000 x g for 10 minutes to dry the membrane.

15. Place the HiBind^? DNA mega column into a new 50 ml  centrifuge tube (not supplied). Add 1.5-3 ml Elution Buffer (10mM  Tris-HCl, pH 8.5) or water to the column. Incubate at room temperature  for 5 minutes.

16. Centrifuge at 5000 x g for 5 minutes to elute the DNA.