• Save 10%
Biomiga

Ultrafast Ultra High Yield Endofree Plasmid Miniprep Kit II, 50 Preps, Biomiga


List price: $80.00  

$72.00

You save: $8.00 (10%)
Biomiga
PD1222-01
In stock
+

Ultrafast Ultra High Yield Endofree Plasmid Miniprep Kit II yields up to 80ug transfection grade DNA in less than 25 min.

Key to the kit is our 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.

Plasmid isolated with traditional protocol normally contains high level of endotoxins (lipopolysaccharides or LPS). For transfection of endotoxin sensitive cell lines or microinjection, the endotoxins should be removed before the applications. The EZgeneTM endofree system uses a specially formulated buffer that extracts the endotoxin from the plasmid DNA. Two rounds of extraction will reduce the endotoxin level to 0.1 EU (Endotoxin) per µg of plasmid DNA. The endofree plasmid miniprep kit provides an efficient endotoxin removal step into the traditional purification procedure to produce transfection grade plasmid DNA.

This kit is designed for fast and efficient purification of plasmid DNA from 3 to 12 mL of E. coli culture. The mini column has a DNA binding capacity of 80 µg.

The purified endofree DNA is ready for downstream applications such as transfection of endotoxin-sensitive cell lines, primary cultured cells or microinjection.

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 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. Please reference Table 2 for theendA information.

Table2 endA strains of E. Coli .

EndA- Strains of E. Coli

DH5?

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.

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

Two endotoxin removal procedures are provided. Protocol A removes endotoxin during the purification of plasmid DNA and Protocol B removes endotoxin after the purification of plasmid DNA.

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: It is stable for half a year under room temperature. Spin down RNase A vial briefly. Add the RNase A solution to buffer A1 and mix well before use.
  • Add 8 mL (PD1222-00) or 60 mL (PD1222-01) or 96 mL (PD1222-02) 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 users

  • 96-100% ethanol.
  • 1.5 mL and 2.0 mL microcentrifuge tubes.
  • High speed microcentrifuge or Vacuum manifold.

Kit Contents

Catalog #

PD1222-00

PD1222-01

PD1222-02

Preps

4

50

250

ezBind Columns

4

50

250

Buffer A1

2.5 mL

25 mL

125 mL

Buffer B1

2.5 mL

25 mL

125 mL

Buffer D1

250 µL

2.5 mL

12.5 mL

Buffer N3

400 µL

5 mL

25 mL

Buffer RET

4 mL

50 mL

250 mL

DNA Wash Buffer*

2 mL

15 mL

3 x 24 mL

Endofree Elution Buffer

1 mL

10 mL

30 mL

RNase A(20 mg/mL)

0.25 mg

(17.5µL)

2.5 mg

(175 µL)

12.5 mg

(625 µL)

User Manual

1

1

1

*Add 8 mL (PD1222-00) or 60 mL (PD1222-01) or 96 mL (PD1222-02) 96-100% ethanol to each DNA Wash Buffer bottle before use.

Safety Information

  • Buffer N3 contain acetic acid, wear gloves and protective eyewear when handling.
  • Buffer N3 and RET 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

pd1220__pd122233521_495_02

None