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Plasmid ezFilter Mega10 Kit

+

Plasmid ezFilter Mega10 Kit
Features :
Price : $140.00
Quantity :
Catalogue#: PD1614-01
  
 

Description :

Introduction

Key to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBindTM matrix while proteins and other contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or elution buffer.

Unlike other procedures, our patented plasmid purification kit has no guanidine salt in the buffer, the purified DNA is guanidine/ion exchange resin residues free which enable the high performance of downstream applications such as transfection, restriction mapping, library screening, sequencing, as well as gene therapy and genetic vaccinations.

Important Notes

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 2 to 3 times. Reference Table 1 for the commonly used plasmids,

Table 1 Commonly used plasmids.

Plasmid

Origin

Copy Numbers

Expected Yield(µg per  500 mL)

pSC101

pSC101

5

50-60

pACYC

P15A

10-12

80-100

pSuperCos

pMB1

10-20

80-150

pBR322

pMB1

15-20

100-150

pGEMR

Muted pMB1

300-400

2000-2500

pBluescriptR

ColE1

300-500

2000-3000

pUC

Muted pMB1

500-700

3000-4000

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. For purifying plasmid DNA from endA+ strains (Table 2), we recommend use product PD1714.  

Table2 endA strains of E. 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.

Culture Volume: Use a flask or tube with a volume at 4 times 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.

Table 3 The optimal cell mass, culture Volume and Binding Capacity for the mega DNA units,

DNA Units

Mega 3

Mega 6

Mega 10

Optimal Cell Mass

1200

2500

4500

Culture Volume

500 mL

1000 mL

1500 mL

Binding Capacity

3-4 mg

6-7 mg

10-12 mg

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 (22-25oC). 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.

Important

  • RNase A: 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.
  • 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.
  • The proper volume of buffer ratio of A1:B1:C1: 100% ethanol =1:1:1.2:1.2.
  • Make sure the availability of centrifuge and vacuum manifold, especially, after mixing the lysate with ethanol, the sample needs to be processed immediately by vacuum.

Materials supplied by users

  • 70% ethanol and 100% ethanol.
  • Pump-driven vacuum system, 500 mL bottle or 1,000 mL bottle (Corning# 430518 or 430282) or equivalent pyrex glass bottles.
  • 50 mL conical tubes.

Kit Contents

Catalog#

PD1614-00

PD1614-01

PD1614-02

Preps

1

2

10

DNA Unit

1

2

10

Filter Unit

1

2

10

Replacement Cup

1

4

20

Buffer A1

110 mL

210 mL

2 x 530 mL

Buffer B1

110 mL

210 mL

2 x 530 mL

Buffer C1

130 mL

250 mL

3 x 450 mL

RNase A (20 mg/mL)

11 mg(550 µL)

21 mg(1.1 mL)

120 mg(4 x1.5 mL)

Elution Buffer

30 mL

60 mL

270 mL

User Manual

1

1

1

 Safety Information

  • Buffer C1 contains acidic acid, wear gloves and protective eyewear when handling.
  • Buffer C1 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

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