96-well Plasmid DNA Isolation Kit, 10x 96 well plate

BP-10 96-Well Spin Column Plate Plasmid DNA Minipreps

(A) Before use, add boiled RNase A to Solution I. Solution I should be stored at 4°C for frequent use, or stored at -20°C if not used for a long period.

(B) Solution II may form a precipitate upon storage. If necessary, dissolve the precipitate by warming at 37°C.

(C) Before use, add 240ml of 96-100% ethanol to 60ml Wash Solution. If volume of Wash Solution is not sufficient as 60ml due to leaking during transportation, it is necessary to re-measure its volume, and determine how many milliliters of ethanol to be added accordingly (volume of added ethanol : volume of Wash Solution = 4:1).

(D) Elution Buffer is 2.5 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or water can be used, yield is generally 20% lower.

Storage: With the exception of the RNase A, the kit may be stored at room temperature. The RNase A should be stored at 4?C. The kit is stable for 12 months at room temperature. For longer storage, keep all contents cold.

Principle

The BP-10 96-well spin column plate plasmid DNA purification kit provides a simple, efficient, and automated high throughput method for Mini plasmid DNA purifications. Plasmid DNA is selectively adsorbed in silica gel-based BP-10 columns in the 96-well plate and other impurities such as proteins, salts, and nucleotides are washed away. Plasmid DNA can be eluted in a small volume of Tris buffer.

Note: This kit is used for preparation of up to 10ug of pure plasmid DNA in each well.

Applications

• Plasimid DNA purification

Features

• Fast - Entire procedure takes 60 minutes.
• Preparation of high quality plasmid DNA from bacterial culture. Purified DNA can be used in any downstream applications such as sequencing, transformation, restriction enzymatic digestion, and ligation.
• High yields (>80%) and reproducible.
• No phenol / chloroform extraction or ethanol precipitation.

Quality Control

All components in the kit are tested in purification of 96 x 10 ug pUC 18 DNA from overnight culture.

Procedure for Purification of Plasmid DNA

1. Fill each well of a Deep Well Collection Plate with 1.3ml of growth medium containing the appropriate selective agent. Inoculate each well from a single bacterial colony. Incubate the cultures for overnight or 20-24 hours at 37°C with shaking at 300 rpm.
2. Harvest the bacterial cells in the Deep Well Collection Plate by centrifugation for 5 min at 1500 rpm in a centrifuge with a rotor for microtiter plates. The Deep Well Collection Plate should be covered with adhesive tape during centrifugation. Remove media by inverting the Deep Well Collection Plate.
   
   1. Note: To remove the media, peel off the tape and quickly invert the Deep Well Collection Plate over a waste container. Tap the inverted Deep Well Collection Plate firmly on a towel to remove any remaining droplets of medium.
3. Resuspend each bacterial cells in the Deep Well Collection Plate in 100ul Solution I. Tape the Deep Well Collection Plate with sealing film and mix by vortexing and keep for 3 minutes.
   
   1. Note: Ensure that RNase A has been added to Solution I. The pelleted cells in the Deep Well Collection Plate be resuspended completely leaving no cell clumps.
4. Add 200ul of Solution II to each well, seal the Deep Well Collection Plate with new tape, mix gently but thoroughly by inverting 10 times, and keep for 2 minutes.
   
   1. Note: Do not vortex at this step, as this may cause shearing of the bacterial genomic DNA. Do not incubate for more than 5 minutes. Additional incubation can result in increased levels of open circular plasmid DNA. At the end of the incubation, the lysate should appear viscous and free of bacterial cell clumps. Avoid extended exposure of Solution II to air since CO₂ can reduce the effectiveness of this solution.
5. Add 350ul Solution III to each well, seal the Deep Well Collection Plate with new tape, and mix immediately by inverting 10 times. Keep for 3 minutes.
   
   1. Note: Gently invert the taped Deep Well Collection Plate 10 times to ensure uniform precipitation.
6. Spin down at 8,000 rpm for 15 minutes.
7. Place a BP-10 96-well spin column plate on the top of a new Deep Well Collection.
8. Transfer the above supernatants (step 6) to BP-10 96 well plate using an 8-channel pipette. Centrifuge at 3,000 rpm for 5 minutes.
9. Discard the flow-through in the Deep Well Collection. Add 500ul of Wash Solution to each well of BP-10 96-well plate, and centrifuge at 6,000 rpm for 5 minutes.
10. Repeat wash procedure in step 9.
11. Discard the flow-through in the deep well collection plate. Spin at 6,000 rpm for additional 5 minutes to remove residual Wash Solution.
12. Place the BP-10 96-well spin column on the top of a 96-well storage plate. Add 50ul of Elution Buffer into the center part of the membrane of each well and incubate at 37- 50°C for 2 minutes. Spin at 6,000 rpm for 5 minutes.
13. Plasmid DNA is ready for use or store at -20°C freezer.
    
    1. Note: It is important to add the Elution Buffer into the center part of each well.

Troubleshooting:

1. RNA contamination
   
   1. RNase A digestion might be insufficient. Check culture volume against recommended volumes, and reduce if necessary. If RNase A solution is kept for more than 6 months, add more RNase A.
   2. Prolong the standing time after adding supernatant to the BP-10 96-well plate.
   3. It is extremely important to add the Elution Buffer to the center of the BP-10 96-well plate. Pre-warming the Elution Buffer to 80?C can improve recovery yield of DNA. Or after adding the Elution Buffer to the BP-10 96-well plate, incubate at 55?C to 60?C for 3?5 minutes.
2. Low yield of plasmid DNA
   
   1. Alkaline lysis was inefficient: If cells have grown to very high densities or a larger amount of cultured medium than recommended was used, the ratio of biomass to lysis reagent is shifted. This may result in poor lysis conditions, because the volumes of solution I, II, and III are inefficient.
      1. Reduce culture volume or increase volumes of Solution I, II and III.