| Semi-automated Method for Fosmid Mini-Preps Using Montage® BAC Miniprep96 Kit on the Beckman Biomek® FX Nucleic Acid Preparation System | Tech Note - TN5000EN00 > Product Catalogue > Request Automation Software | |
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Introduction
Genome sequencing has been accelerated forward by the use of cloning large fragments of genomic DNA into fosmids. These cloned pieces of DNA are usually up to 40 kb in size, whereas the fosmid vectors themselves are predominantly around 9 kb. Despite the challenges of low vector copy number, the need for high throughput automation of fosmid template preparation, like BAC’s, is significant. Here we describe a semi-automated preparation method using the Montage BAC96 Miniprep Kit on the Biomek FX workstation. Vacuum filtration and shaker mixing steps were performed off-line, however, these can be carried out on the FX utilizing SPE and orbital shaker ALP’s, thus supporting a fully automated procedure.
The protocol was developed to maximize yields, decrease well-to-well variability, and provide high quality DNA sequence data. Culture and lysis conditions were optimized to accomplish this. The protocol, which relies primarily on vacuum filtration for sample processing, takes approximately 1 hour.
Configuration of the Biomek FX Deck Setup
Prior to starting:
Make sure deck is set up as follows:
P2: P200 Tips for alkaline lysis
P3: P200 Tips for resuspension
P4: Tip wash reservoir
P5: Solution 1 reservoir
P6: V-bottom storage plate
P7: Wash solution reservoir
P8: Solution 2 reservoir
P9: Deep well block with pelleted cells
P10: Resuspension solution reservoir
P11: Solution 3 reservoir
P12: MultiScreen96 CLEARING or BAC plates
Procedure:
Growth and Cell Pelleting
1. Prepare fosmid precultures from colonies or glycerol stocks by inoculation into 1.0 mL aliquots of 2 x LB plus antibiotic (e.g., 12µg/mL chloramphenicol) in 96 well culturing blocks (2.2 mL capacity). Cover blocks with foil sealing tape and pierce prior to securing in an incubator/shaker. Incubate at 37 °C and 320 rpm for 18 hours. Preparing precultures for seeding into cultures helps to normalize yields from well to well. This normalization of yields aids in preparation for downstream applications.
NOTE: 96 well blocks for growing 1.0 mL precultures are not included in this kit but may be ordered separately (Millipore Cat. No. LSKC CB0 50). Foil sealing tape is also not included.
2. Inoculate 3 µL of fosmid precultures into 1.5 mL aliquots of 2 x LB plus antibiotic (e.g., 12 µg/mL chloramphenicol) in 96 well culturing blocks (2.2 mL capacity). Cover blocks with foil sealing tape and pierce prior to securing in an incubator/shaker. Incubate at 37 °C at 320 rpm for 17–20 hours (OD650 reading should be in the range of 3.0–4.0).
3. Centrifuge at 1500xg for 5 minutes. Decant culture supernatant to a container for proper disposal.
4. Place deep well block with pellets on the Biomek FX in position P9.
Fosmid Purification
5. Start program.
6. Add 100µl of Solution 1 (P5) to the deep well block (P9) and wash tips (P4). Manually move block to shaker off-line and agitate at 800 rpm for 5 minutes.
7. Add 100µl of Solution 2 (P8) to the deep well block (P9) and wash tips (P4). Manually move block to shaker off-line and agitate at 800 rpm for 1 minute.
8. Incubate for 2 minutes at room temperature.
9. Add 100µl of Solution 3 (P11) to the deep well block (P9). Manually move block to shaker off-line and agitate at 800 rpm for 3 minutes.
10. Transfer the lysates from the deep well block (P9) to the CLEARING plate (P12). Unload lysate tips.
11. Filter CLEARING plate off-line at 8 inches Hg for approximately 10 minutes or until all wells are empty and collect the cleared lysates into the BAC plate.
12. Discard the CLEARING plate, move the BAC plate to the top of the vacuum manifold, and filter at 24 inches Hg for 8 minutes or until all wells are empty.
13. Place BAC plate in position P12, load resuspension tips (P3), and add 200µl of wash solution (P7) to the BAC plate.
14. Filter at 24 inches Hg for approximately 5 minutes or until all wells are empty.
15. Replace BAC plate to position P12 and add 30µl of resuspension solution (P10) to the BAC plate. Manually move plate to shaker off-line and agitate at 800 rpm for 10 minutes.
16. Replace BAC plate to position P12 and transfer purified fosmid samples from BAC plate to the V-bottom storage plate (P6).
Table 1a. Fosmid Yields
Average: | 0.42 µg |
Std Dev: | 0.08 µg |
CV: | 20.2% |
Min: | 0.23 µg |
Max: | 0.60 µg |
Table 1b. Sequencing Reaction Setup
*The annealing temperature was optimized for use with a T7 primer (5'-TAA TAC GAC TCA CTA TAG GG-3') and the vector pCC1FOSä. Optimization of annealing temperature will be required when other vectors and/or primer combinations are employed.
Table 1c. Sequencing Data
Average: | 656 |
Std Dev: | 133 |
CV: | 20.2% |
Pass rate (>300): | 95% |
n | 96 |
Figure 1. Typical Electropherogram
Montage-SEQ384 purified ¼x BigDye Terminator sequencing reaction from a fosmid template run on an ABI 3700. Phred 20 = 683
Conclusion:
Using the Millipore Montage BAC Miniprep96 kit for fosmids results in highly purified DNA which can be used in downstream applications such as sequencing, as was demonstrated here. The DNA yields, under these conditions, were more than adequate for obtaining superior sequence reads. The protocol is reliable and can be fully automated by incorporating a shaker ALP and performing vacuum filtration steps on the Biomek FX.
Ordering information:
Product Name | Millipore Part Number | Qty/Pack |
Montage BAC MiniPrep96 Kit | LSKB 096 01 | 1 X 96 well |
LSKB 096 04 | 4 X 96 well | |
LSKB 096 24 | 24 X 96 well | |
Millipore Vacuum Manifold | MAVM 096 0R | |
Acknowledgement: Millipore would like to thank Genevieve Di Bartolo and the Joint Genome Institute for the use of their clones.
Biomek is a trademark of Beckman Instruments, Inc.
pCC1FOS is a trademark of Epicentre Technologies or an affiliated company.
SYBR is a registered trademark of Molecular Probes, Inc.
Millipore and Montage are registered trademarks of Millipore Corporation.
ABI Prism is a trademark of Applera Corp.
BigDye is a trademark of PE Corporation.
Copyright © 2003 Millipore Corporation, Billerica, MA