Plasmid Miniprep Kit I, Biomiga

Biomiga's Plasmid Purification System

·         Superior Purity:This patented Plasmid Purification system uses no chaptropic salts in the buffer, and the resulting purified DNA is free of guanidine salts, cesium chloride, ethidium bromide and ion exchange resin residues, which enable the highest performance of downstream applications including gene therapy and genetic vaccination.

·         Ultra High Yield:When comparing Biomiga' Plasmid Purification System to other purification methods, our methodology produces higher DNA yield while requiring fewer bacterial cultures.

·         Time Saving:Using our unique Megaprep Kit, 10 mg plasmid DNA can be purified from 1,000 mL culture within 1 hour!

·         Environment benefit:The EZgene System is the only method available that does not utilize Guanidine Salt, a hazardous protein denaturant, in the silica based purification system, which will ensure the safety and health of scientists and the environment!

      For a complete list of Biomiga’s Plasmid Purification Systems, Click here

 

Citations

Murine Xenograft Model for Human Uterine Fibroids: An In Vivo Imaging Approach.  Reproductive Sciences, 2009, 16(9), 827-842

Characterization of monoclonal antibodies against goat interleukin-18 and their application in the measurement of goat interleukin-18 in LPS-stimulated peripheral blood mononuclear cells by sandwich ELISA. Veterinary Immunology and Immunopathology, Volume 138, Issue 3, 2010, 235-238

Platelet-Derived Growth Factor C Is Upregulated in Human Uterine Fibroids and Regulates Uterine Smooth Muscle Cell Growth1. Biology of Reproduction, 2009, vol. 81 no. 4749-758

Cloning, characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[?]pyrene exposure. Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology , 2010,Volume 152, Issue 2, August 2010, Pages 160-166

An Mpeg (macrophage expressed gene) from the Pacific oyster Crassostrea gigas: Molecular characterization and gene expression.Fish & Shellfish Immunology, 2011, Volume 30, Issue 3, March 2011, 870-876

 

Plasmid Miniprep Kit I, Biomiga

The key to Plasmid Miniprep Kit I is Biomiga's proprietary DNA binding systems that allow the high efficient reversible binding of DNA to the mini column while proteins and other impurities are removed by wash buffer. Nucleic acids are then eluted with sterile water or elution buffer.

Biomiga's Plasmid Miniprep Kit I is designed for fast and efficient purification of plasmid DNA from 1 to 4 mL of E. coli culture. The mini column has a plasmid DNA binding capacity of 50 µg. The yield from 1 mL culture is typically around 8 to 12 µg. Biomiga's Plasmid Miniprep Kit II (PD1213), with the plasmid DNA binding capacity of 80 µg, is recommended if higher yield (>50 µg) is desired.

The purified DNA is ready for downstream applications such as cloning/subcloning, RFLP, sequencing, and transfection of robust cells such as HEK293 cells.

 

Plasmid Copy Numbers and Yield

Plasmid Copy Numbers: The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid. The protocols are optimized for high copy number plasmid purification. For low copy number plasmids, both the culture volume and the buffer volume need to be scaled up 3 to 5 times. Please reference Table 1 for the commonly used plasmids,

Table 1 commonly used plasmid and expected yield.

Plasmid

Origin

Copy Numbers

Expected Yield

(µg per 1 mL)

pSC101

pSC101

5

0.1-0.2

pACYC

P15A

10-12

0.4-0.6

pSuperCos

pMB1

10-20

0.4-1

pBR322

pMB1

15-20

0.6-1

pGEMR

Muted pMB1

300-400

6-7

pBluescriptR

ColE1

300-500

6-8

pUC

Muted pMB1

500-700

8-12

 

Host Strains: The strains used for propagating plasmid have significant influence on yield. Host strains such as Top 10 and DH5a yield high-quality plasmid DNA. endA+ strains such as JM101, JM110, HB101, TG1 and their derivatives, normally have low plasmid yield due to either endogenous endonucleases or high carbohydrates released during lysis. We recommend transform plasmid to an endA- strain if the yield is not satisfactory.

Table2 endA- strains ofE. Coli.

EndA- Strains of E. Coli

DH5α

DH1

DH21

JM106

JM109

SK2267

SRB

XLO

TOP10

DH10B

JM103

JM107

SK1590

MM294

Stbl2TM

XL1-Blue

BJ5182

DH20

JM105

JM108

SK1592

Select96TM

Stbl4TM

XL10-Gold

EndA+ Strains of E. Coli

C600

JM110

RR1

ABLE® C

CJ236

KW251

P2392

BL21(DE3)

HB101

TG1

TB1

ABLE® K

DH12STM

LE392

PR700

BL21(DE3)pLysS

JM101

JM83

TKB1

HMS174

ES1301

M1061

Q358

BMH 71-18

All NM  strains

All Y strains

 

Optimal Cell Mass (OD600 x mL of Culture): This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) for 12 -16 hours to a density of OD600 2.0 to 3.0. If rich medium such as TB or 2xYT are used, make sure the cell density doesn't exceed 3.0 (OD600). A high ratio of biomass over lysis buffers result in low DNA yield and purity. The mini column has an optimal biomass of 10-15. For example, if the OD600 is 3.0, the optimal culture volume should be 1-5 mL. For over amount of cell numbers, either reduce the biomass or scale up the volumes of Buffer A1, B1 and N1.

Culture Volume: Use a flask or tube 4 times bigger in volume than the culture medium to secure optimal condition for bacteria growth. Don't exceed the maximum culture volume suggested in the protocol. Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity.

 

Storage and Stability

Buffer A1 should be stored at 4°C once RNase A is added. All other materials can be stored at room temperature (22-25°C). The Guaranteed shelf life is 12 months from the date of purchase.

 

Before Starting

Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps and pay special attention to the followings,

 

Important

  • RNase A: 20 mg/mL. It is stable for more than half a year when stored at room temperature. Spin down RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use. Store at 4°C.

  • Add 8 mL (PD1211-00) or 60 mL (PD1211-01) or 96 mL (PD1211-02) or 60 mL (PD1211-03) 96-100% ethanol to each DNA Wash Buffer bottle before use.

  • Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50°C to dissolve the precipitates before use.

  • Keep the cap tightly closed for Buffer B1 after use.

  • Ensure the availability of centrifuge capable of 13,000 rpm.

  • Carry out all centrifugations at room temperature.

Materials supplied by user

  • High speed microcentrifuge or Vacuum manifold.

  • 96-100% ethanol.

  • 1.5 mL microcentrifuge tubes

 

Kit Contents

Product Code

PD1211-00

PD1211-01

PD1211-02

Preps

4

50

250

ezBind Columns

4

50

250

Buffer A1

1.2 mL

15 mL

70 mL

Buffer B1

1.2 mL

15 mL

70 mL

Buffer N1

1.6 mL

20 mL

100 mL

Buffer KB

3 mL

30 mL

135 mL

DNA Wash Buffer*

2 mL

15 mL

3 x 24 mL

Elution Buffer

600 µL

10 mL

30 mL

RNase A (20 mg/mL)

0.2 mg

(10 µL)

1.5 mg

(75 µL)

7.0 mg

(350 µL)

User Manual

1

1

1

*Add 60 mL (PD1211-01) or 96 mL (PD1211-02) or 60 mL (PD1211-03) 96-100% ethanol to each DNA Wash Buffer bottle before use.

 

Safety Information

  • Buffer N1 contains acidic acid, wear gloves and protective eyewear when handling.

  • Buffer N1 and KB contains chaotropic salts, which may form reactive compounds when combines with bleach. Do not add bleach or acidic solutions directly to the preparation waste.

 

Operating Protocol