Protocol for bacmax96™ dna purification kit

BACMAX96™ DNA Purification Kit
The BACMAX96™ DNA Purification Kit is de-
signed for easy, reliable isolation of high-quality
BACMAX96™ DNA Purification Kit
BAC and fosmid DNA in a 96-well format. Small- Contents
er batches of cultures can be prepped using The BACMAX96™ DNA Purification Kit con- smaller portions of the 96-well plates.
tains sufficient reagents to perform four 96-well plate purifications.
The protocol is based on a modified alkaline- Cat. Nos. BAC96066 and BAC96116 both
lysis procedure that incorporates proprietary en-zyme technologies into an easy-to-use format.
contain the following:
Purification starts with cell pellets obtained by centrifugation of 1.2 ml E. coli cultures grown in a 96-well plate. Lysates are cleared in the pres- efficiently removes unwanted RNA. Following fil- tration and selective precipitation steps, Plas-mid-Safe™ ATP-Dependent DNase is used to remove any remaining bacterial genomic DNA.
To maximize flexibility, culture blocks and filtra-tion plates can be supplied by the user (Cat.
No. BAC96066) or can be purchased with the kit (Cat. No. BAC96116). Filtration steps can therefore be performed via vacuum or centrifu- gation, making the kit easy to use with standardlaboratory equipment. The kit is also amenable to semi- or fully automated purification systems.
Cat. No. BAC96116 also contains:
The BACMAX96 Kit consistently yields up to 900 ng of DNA from a single-copy BAC cloneand up to 2.5 µ g of DNA from CopyControl™ BAC clones1 induced to high-copy number. The unprecedented yields from CopyControl clonesis due in part to an autoinduction protocol that was specifically designed for a high-throughput format. In this “hands-off” approach, the Autoin-duction Solution is added to the growth media * BACMAX96™ Solution 2 may form a precipitate prior to inoculation and copy-number remains during storage. If this occurs, heat the bottle suppressed until shortly before the cells are har- at 37oC until the precipitate dissolves. Mix thoroughly and cool to room temperature. Product Specifications
Related Products: The following products are
also available:
Storage: Store the BACMAX96 Solutions 1, 2
and 3 at room temperature. Store the remain-
der of the kit components at -20oC in a freezer − CopyControl™ Fosmid Library Production Kit − BAC-Tracker™ Supercoiled DNA Ladder Quality Control: The BACMAX96 DNA Purifica-
tion Kit is function-tested by purifying a 130 kb BAC. DNA quality and yield are assayed by gelelectrophoresis, fluorimetry and restriction en- Reference:
1. EPICENTRE Forum (2002) 9 (1), 4.
www.EpiBio.com • (800) 284-8474 • (608) 258-3080 • Fax (608) 258-3088
EPICENTRE
BACMAX96™ DNA Purification Kit
Figure 1. An overview of the BACMAX96™ DNA Purification Kit protocol.
Grow BAC or fosmid clones (with or without
Autoinduction Solution in a deep-well 96-well plate
Harvest the cells
Resuspend, lyse and neutralize using
BACMAX96™ Solutions 1, 2 and 3
(BACMAX96 RNase Blend added to Solution 1)
Transfer supernatant to filter plates
Collect cleared lysate in receiver plate
Precipitate nucleic acid with ethanol
Wash with 70% ethanol and air dry
Resuspend nucleic acid and treat with Plasmid-Safe™ ATP-Dependent
DNase to remove chromosomal background
Heat inactivate the Plasmid-Safe™ ATP-Dependent
DNase and resuspend overnight
EPICENTRE
BACMAX96™ DNA Purification Kit
BACMAX96 DNA Purification Protocols
The following equipment is not supplied with the BACMAX96 kit (BAC96066** or BAC96116):
** If the standard BACMAX96 Kit (BAC96066) is used, appropriate growth, collection and filtration plates will need to be supplied as well.
A. Growing 96-Well Bacterial Cultures
1. Single-copy BAC and fosmid clones: Dispense 1.2 ml of LB containing the appropriate antibiotic into each well of a 2 ml deep well culture plate.
CopyControl BAC clones: Dispense 1.2 ml of LB containing chloramphenicol (12.5 mg/ml) andBAC Autoinduction Solution (6 ml/ml media) into each well of a 2 ml deep well culture plate.
CopyControl Fosmid clones: Dispense 1.2 ml of LB containing chloramphenicol (12.5 mg/ml) andFosmid Autoinduction Solution (2 ml/ml media) into each well of a 2 ml deep well culture plate.
2. Inoculate each well with an isolated colony from a freshly streaked plate using a toothpick or from a glycerol stock using a 96-pin device or multi-channel pipette.
3. Seal the plate using an air permeable plate seal. Incubate cultures in an incubator/air shaker for approximately 17 hours at 37oC with constant shaking at 250 rpm.
B. Before Starting
1. Required plastics (per 96 purifications) Listed items are supplied in the BAC96116 Kit.
In addition, 6 reservoir trays will be needed for each time this protocol is used, regardless of thenumber of purifications performed.
2. The table below lists the recommended volumes of BACMAX96 Solutions 1, 2 and 3 for purifica- tions from single or multiple 96-well plates. Keep BACMAX96 Solutions 1 and 3 on ice and
BACMAX96 Solution 2 at room temperature.
3. Just prior to use: add 30 ml of BACMAX96 RNase Blend per 1 ml of BACMAX96 Solution 1 to be EPICENTRE
BACMAX96™ DNA Purification Kit
C. BAC/Fosmid Purification Protocol
1. Pellet the cells at 1,000 x g for 10 minutes at 4oC in a table top centrifuge. Discard the plate sealer and carefully decant the media. Turn the plate up side down on a stack of paper towels todrain residual media.
2. Add 100 µl of chilled BACMAX96 Solution 1 to each well of the culture plate. Cover the culture plate using an adhesive plate sealer and press firmly with your fingertips to ensure the wells aretightly sealed.
3. Mix by vortexing the plate at maximum speed. Make sure the pellets are completely resuspended 4. Carefully remove the plate sealer and discard. Add 200 µl of room temperature BACMAX96 Solution 2 to each well of the culture plate. Cover the culture plate securely using a new platesealer. Mix by inverting the plates 2-3 times very gently.
5. Incubate at room temperature for 4 minutes. Note: The lysis reaction should not exceed 5 min- utes. Carefully remove the plate sealer and discard. To ensure there will be no cross contamina-tion between adjacent wells, remove any excess liquid by blotting the plate with a paper towel.
6. Add 150 µl of chilled BACMAX96 Solution 3 to each well of the culture plate. Cover the culture plate securely using a new plate sealer. Mix by inverting the plates 2-3 times very gently.
8. Centrifuge the block at ≥1,400 x g for 10 minutes at 4oC.
9. Prior to transferring the lysate, place the filter plate on top of the collection plate. Transfer 300 µl of the lysate from the culture plate to the corresponding wells of the filter plate. When filtering thelysate via centrifugation, spin the filter and collection plate assembly at 1,000 x g for 5 minutes at4oC. If you are using a vacuum manifold, follow the manufacturer’s recommendations.
10. Add 2 volumes of absolute ethanol (200 proof) to the recovered lysate. Cover the receiver plate securely using a new plate sealer. Mix by inverting the plates 5-6 times.
11. Centrifuge the plate at ≥1,400 x g for 20 minutes at 4oC to precipitate the DNA.
12. Carefully remove the plate sealer and discard. Pour off the ethanol and remove any excess liquid by placing the plate up side down on a clean stack of paper towels.
13. Add 600 µl of freshly prepared 70% ethanol to each well. Cover the plate with a fresh plate sealer, and centrifuge the plate at ≥1,400 x g for 10 minutes at 4oC.
14. Remove the plate sealer and pour off the ethanol. Remove any excess liquid by placing the plate up side down on a clean stack of paper towels and tapping several times. Turn the plateright side up and air dry at room temperature for 30 minutes.
15. During this drying step, prepare the appropriate amount of Plasmid-Safe Master Mix (use the EPICENTRE
BACMAX96™ DNA Purification Kit
16. Add 20 µl of TE Buffer (sterile deionized water or Tris-buffer can also be used). Cover the re- ceiver plate securely using a new plate sealer. Place the plate on a rotary shaker and shake verygently (lowest speed) for 5 minutes. Give the plate a 1 minute quick spin to collect any liquid notat the bottom of the wells.
Plasmid-Safe Master Mix: The table below lists the reagent volumes required to make enoughmaster mix for single to multiple 96-well plates.
17. Remove and save the plate sealer. Add 4 µl of the Plasmid-Safe Master Mix to each well and
cover the receiver plate securely using the saved plate sealer. Mix by swirling the plate or placethe plate on a rotary shaker and shake very gently (lowest speed) for 2-3 minutes.
18. Centrifuge the plate at ≥1,400 x g for 1 minute at 4oC.
19. Incubate the plate at 37oC for 20 minutes.
20. Incubate the plate at 65oC for 15 minutes to inactivate the enzyme.
21. Cool the plate at room temperature. Centrifuge the plate at ≥1,400 x g for 1 minute at 4oC to col- lect any condensation that may have formed.
22. The DNA is now ready for use. We suggest storage at 4oC if the DNA is to be used in the next 48 hours, otherwise store the DNA at -20oC.
D. Expected Yields
Single-copy BAC and fosmid clones: Yields range from 500-900 ng of DNA depending on the growthcharacteristics and size of the construct. We recommend 8 µl of sample for each end sequencingreaction or restriction enzyme digest.
CopyControl BAC and fosmid clones: Yields range from 2.1-2.5 µg of DNA depending on the growthcharacteristics and size of the construct. As little as 1 µl of sample can be used for each end se-quencing reaction or restriction enzyme digest.
BACMAX96, Plasmid-Safe, CopyControl, BAC-Tracker, BACMAX, Fast-Link and End-It are trademarks of

Source: http://www.ecogen.es/upfiles/A37441.pdf