EasyPure Genomic DNA Kit

タイトルまたは説明

Cat.No.  EE**1

Ordering Information:

Storage:RNase A and Proteinase K solutions at **0°C for one year; other components at room temperature (****5°C) for one year.

Description

EasyPure® Genomic DNA Kit provides a simple and convenient way to isolate high quality genomic DNA from a variety of mammalian cells, tissues, E.coli and yeast. Cells and tissues are enzymatically lysed. DNA is bound to silica-based column. The isolated DNA is suitable for PCR, restriction enzyme digestion and southern blotting.

• DNA yield up to *5 μg.

• Complete removal of contaminants and inhibitors.

• DNA range of ****0 kb.

• Column based purification, no organic extraction or ethanol precipitation.  

Kit Contents

Procedures

Before starting, adding appropriate volume of *****0% ethanol to WB2.

1. Preparing materials

• Mammalian Cells

a) Adherent cells: Remove the culture media from culture plate and harvest cells by trypisin or other methods. Collect cells by centrifuging at **0×g for 5 minutes, and then remove the supernatant .

b) Suspension cells: Harvest cells by centrifuging at **0×g for 5 minutes. Remove the supernatant.

c) Add **0 μl LB2 to the cell pellet, mix thoroughly by vortexing or pipetting.

Optional: If RNA-free genomic DNA is required, add *0 μl RNase A to the lysate.

d) Add *0 μl Proteinase K to the lysate. Mix the tube briefly by vortexing, then incubate at room temperature for 2 minutes.

• Mammalian Tissues (Prepare *5°C water bath or heater before starting).

a) Transfer ≤*5 mg (spleen≤*0 mg) chopped tissue to a sterile 1.5 ml microcentrifuge tube.

b) Add **0 μl LB2 and *0 μl Proteinase K to the tube. Make sure that the tissue is completely immersed in the tube.

c) Incubate at *5°C until the sample is completely lysed (3 hours are needed for most tissues; **8 hours or longer are needed for mouse tail; mix the lysate 2~3 times every hour).

Optional: If RNA-free genomic DNA is required, add *0 μl RNase A to the lysate, incubate at room temperature for 2 minutes.

d) Centrifuge at *2,**0×g for 5 minutes, transfer the supernatant to a sterile 1.5 ml microcentrifuge tube.

• E.coli Cells (Prepare *5°C water bath or heater before starting).

a) Transfer 1~5 ml cell culture (≤2×**9) to a 1.5 ml tube and centrifuge the tube at *2,**0×g for 1 minute. Discard the supernatant completely.

b) Add **0 μl LB2 and *0 μl Proteinase K into the tube. Resuspend the cell pellet by vortexing or pipetting. c) Incubate at *5°C for *5 minutes.

Optional: If RNA-free genomic DNA is required, add *0 μl RNase A to the sample, incubate at room temperature for 2 minutes.

 • Yeast Cells (Prepare *7°C, *5°C water bath or heater before starting)

Prepare fresh D-glucitol buffer (1 M sorbitol, *0 mM EDTA, *4 mM β-mercaptoethanol). Prepare lyticase. a) Harvest yeast cells (≤5×**7 ) by centrifuging at *2,**0×g for 1 minute. Discard the supernatant.

b) Add **0 μl D-glucitol buffer, *5 units lyticase to the pellet. Mix thoroughly and incubate at *7°C for 1 hour.

c) Centrifuge at 5,**0×g for *0 minutes. Discard the supernatant.

d) Resuspend pellets in **0 μl LB2 and *0 μl Proteinase K, mix thoroughly by votexing.

e) Incubate at *5°C for *5 minutes. Optional: If RNA-free total DNA is required, add *0 μl RNase A to the lysate, incubate at room temperature for 2 minutes.

f) Centrifuge at *2,**0×g for 5 minutes and transfer the supernatant to a sterile 1.5 ml microcentrifuge tube. 2. Add **0 μl BB2, mix by vortexing for 5 seconds, incubate at room temperature for *0 minutes.

3. Centrifuge the tube briefly and transfer all the lysate to a spin column. Centrifuge at *2,**0×g for *0 seconds. Discard the flow through.

4. Add **0 μl CB2, Centrifuge at *2,**0×g for *0 seconds. Discard the flow through.

5. Repeat step 4. 6

. Add **0 μl WB2 (check to ensure you have added ethanol) and centrifuge at *2,**0×g for *0 seconds. Discard the flow through.

7. Repeat step 6.

8. Centrifuge the empty column at maximum speed (≥*2,**0×g) for 2 minutes to remove residual WB2.

9. Place the spin column in a sterile 1.5 ml microcentrifuge tube. Add *****0 μl of Elution Buffer (preheated to *5°C) or sterile, distilled water (pH >7.0, preheated to *5°C) to the column matrix. Incubate at room temperature for 1 minute. Centrifuge at *2,**0×g for 1 minute to elute the isolated genomic DNA. Optional: To get more DNA by repeating step 9.

*0. Store the isolated DNA at **0°C.

Notes

• Do not use too many starting materials in case it affects the extraction performance

• Cut the tissue as small pieces as possible, in case it affects lysis. After complete lysis, the lysate presents sticky appearance, rather than gelatinous appearance.

• To ensure the quality of extracted DNA, use fresh material and avoid repeated freezing and thawing . DNA quality depends on the types of material and storage time.

• Use sterile tubes and pipette tips to avoid the contamination from DNase.

• You may perform the second elution step using the same microcentrifuge tube or different tubes.

CITATIONS

Geng J, et al. ***3. Efficient Attenuation of NK Cell–Mediated Liver Injury through Genetically Manipulating Multiple Immunogenes by Using a Liver-Directed Vector. **0(9):*****9. J Immunol. IF=5.*2. PMID: *******2.

Xie K, et al. ***3. OxyR, an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis GP*2. **8(*0):******3.  Microbiol Res. IF=1.**3. PMID: *******5.

Liu Q. et al. ***2. Identification of the bacteriocin subtilosin A and loss of purL results in its high-level production in Bacillus amyloliquefaciens.. **3(**7):****8. IF= 2.**3.  PMID: *******3.

Xu X. et al. ***2.Hypoxia induces downregulation of soluble guanylyl cyclase β1 by miR**4c*5p. **3(**7):****8. IF= 6.**1.  PMID: *******7.