eLabNotebook - Millipore's Semi-automated Method for Fosmid Mini-Preps Using Montáge* BAC Miniprep96 Kit on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > Genomic DNA Purification > Millipore Fosmid
Mini-Preps Biomek® FX

Millipore's Semi-automated Method for Fosmid Mini-Preps Using Montáge* BAC Miniprep96 Kit on the Beckman Biomek® FX Nucleic Acid Preparation System

Millipore– www.millipore.com

Figure 1. Configuration of the Biomek® FX Deck

(Click to Enlarge)

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

This plate represented 96 different fosmid clones which were prepared following the protocol described above. Yields were calculated using a fluorometric assay with SYBR* Green I nucleic acid gel stain (Molecular Probes, Inc). Plate average, standard deviation, and coefficient of variation are noted, as well as the minimum and maximum yields.

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 pCC1FOS?. Optimization of annealing temperature will be required when other vectors and/or primer combinations are employed.

Table 1c. Sequencing Data

One plate of BigDye reactions with fosmid templates was sequenced and subsequently purified on a Montage-SEQ384 plate. BigDye reactions were injected at 2kV for 15 seconds and run at 6.5kV for 117 minutes. Table 1c shows the q-20 quality scores of the sequence reads determined by the software program Phred. Phred 20 scores correspond to a confidence level of 99.9% accuracy of base calling. Plate average, standard deviation, and coefficient of variation are noted, as well as the pass rate and sample number.

Figure 2. 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.

* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.