质粒小量提取试剂盒
产品名称： 质粒小量提取试剂盒
英文名称： Plasmid MiniPrep Kit
产品编号： 121131-1
产品价格： 0
产品产地： 美国
品牌商标： Gragen
更新时间： null
使用范围： null
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Gragen Plasmid MiniPrep Kit
Catalog No. : 121131
Introduction
The Gragen Plasmid Miniprep Kit isolates plasmid DNA from 1-5 mL cultures of E. coli by using silica membrane. Purified DNA can be directly used for most downstream applications, including automated fluorescent DNA sequencing, restriction endonuclease digestion, and other manipulations.
Components
|
Catalog No. |
Components |
50T |
Storage |
|
121132 |
Resuspension Buffer (S1) |
15 ml |
4oC |
|
121133 |
Lysis Solution (S2) |
20 ml |
15-25oC |
|
121134 |
Binding Solution (S3) |
15 ml |
15-25oC |
|
121135 |
Wash Buffer (W4) |
10 ml |
15-25oC |
|
121136 |
Elution Buffer (E5) |
5 ml |
15-25oC |
|
121137 |
RNase A (10 mg/ml) |
150 μl |
-20oC |
|
121138 |
Spin Columns |
50 sets |
15-25oC |
Protocol
*Add 150μl RNase A to Resuspension Buffer (S1) , mix well and store at 4°C.
*Add 40 ml Ethanol to 10 ml Wash buffer W4 (100T) before use.
1. Transfer 1-1.5 ml cultured bacteria to 1.5 ml microcentrifuge tube and centrifuge at 12, 000 rpm for 30 seconds. Remove and discard the supernatant.
2. Resuspend the bacterial pellet by adding 250 μl Resuspension Buffer (S1) (RN ase A added) and vortexing (or pipetting up and down) well. No bacterial clumps should be visible after resuspension of the pellets.
3. Add 350 μl blue Lysis Solution (S2) and gently mix by inverting and rotating tube several times to obtain a clear blue lysate. A 2-3 minute incubation period may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity.( Do not allow the lysis reaction to proceed more than 5 min.)
4. Add 250 μl yellow Binding Solution (S3) and mix immediately by inverting several times until a loose yellow precipitate forms.
5. Centrifuge at 12,000 rpm (>14, 000 ×g) for 5 minutes until a compact pellet forms.
6. Transfer the supernatant to the Spin column and centrifuge for 1min at 12，000 rpm. Discard all flow-through.
7. Add 650 μl Wash Buffer (W4) to the spin column and centrifuge for 1min at 12,000 rpm. Discard the flow-through. Repeat this step once.
8. Centrifuge the spin column for additional 1 minute at 12,000 rpm to remove residual liquid and transfer the spin column to a sterile 1.5 ml microcentrifuge tube.
9. Add 30-50 μl Elution Buffer (E5) to the spin column and let the column stand for 1 minute at room temperature.
10. Centrifuge for 1 min at 12,000 rpm to elute plasmid DNA. The plasmid DNA can be used directly or stored at -20 °C.
Note:
* Some precipitate may form in Lysis Buffer and Neutralization Buffer after long periods of storage. Dissolve the precipitate by warming in the 37°C water bath
* Please do not vortex Lysis Solution acutely after the addition of Lysis Solution to avoid contamination by genomic DNA.
* Please tightly cap Lysis Solution when not in use to avoid acidification of Lysis Solution from CO2 in the air.