The EZgene high-capacity-plasmid DNA Isolation Kit is designed for rapid purification of BAC, PAC, cosmid and P1 from small volume of bacterial cultures. It is based on a modified alkaline lysis procedure that is specially adapted for spin column. The procedure associated with this kit has been tested using a variety of low copy cosmid, BAC, PAC and P1 in different E. coli strains. In addition, this kit can also be used for high copy plasmid isolation.
Storage and Stability
All EZgeneTM BAC/PAC DNA Isolation Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: Buffer X1/RNase A mixture at 4°C, and all other materials at 22-25°C.
Kit Contents
Catalog#
BMG-PD1311-01
BMG-PD1311-02
Preps
50
250
ezBindTM Columns
50
250
Buffer X1
20 mL
90 mL
Buffer X2
20 mL
90 mL
Buffer X3
20 mL
90 mL
BAC Binding Buffer
2 mL
10 mL
Elution Buffer
10 mL
30 mL
DNA Wash Buffer
12 mL
50 mL
RNase A
100 µL
500 µL
User Manual
1
1
Important:
Expected yield: The yield of BAC is around 0.6 µg from 2 mL LB culture and 1 µg from 5 mL culture. If cultured in TB, the yield is about 1 µg from 1.5 mL culture and 3 µg from 5 mL culture
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.
Materials required but not supplied
Microcentrifuge capable of at least 12,000 x g
Microcentrifuge set at 4°C capable of at least 12,000 x g
Sterile Deionized Water
Sterile 1.5 mL and 2.0 mL Centrifuge Tubes
10-15 mL Culture Tubes
96-100% Ethanol
96%-100% Isopropanol
Before start
Add whole vial of RNase A to Buffer X1 and Store at 4°C.
Dilute DNA Wash Buffer with absolute ethanol as follows,
PD1311-01 Add 48 mL ethanol to the bottle.
PD1311-02 Add 200 mL ethanol to each bottle.
Dilute BAC Binding Buffer with isopropanol as follows,
PD1311-01 Add 25 mL isopropanol to the bottle.
PD1311-02 Add 125 mL isopropanol to each bottle.
It’s strongly recommended to use 2 x YT media for the cultivation of cosmids, BACs, PACs, and P1s.
Buffer X2 should be kept at room temperature. Check for SDS precipitation before use. If necessary re-dissolve SDS precipitate by warming. Close the Buffer X2 bottle immediately after use to avoid acidification that may result from air CO2.
Chill Buffer X3 for precipitation enhancement.
Prewarm dd H2O or elution buffer at 60°C before elution.
Use 4°C microcentrifuge for step 6.
