Preparation of DNA Template For Direct Sequencing of Large Insert PAC and BAC Plasmid

This protocol is designed to allow the use of 96-well trays (8x12 microtiter tray format) and multi-channel devices from the initial inoculation step to the final cycle sequence reaction.  It will yield approximately 200ng of purified plasmid per ml of culture fluid.  When using one of the accompanying sequencing methods, one cycle sequence reaction will require 160-300ng of template.

 1. Streak clone stock to single colony on LB (+antibiotic) plate. Inoculate 1ml LB (+antibiotic) culture with single colony, grow overnight at 37⁰C. Using 3 ul of (overnight growth) single colony culture fluid, inoculate a single or multiple 96 deep-well blocks containing 1.2-1.6 ml per well of LB media with appropriate antibiotic. Alternatively, 24 deep-well or 48 deep-well blocks may be used with up to 4.8 or 2.4ml of media respectively. Cover each block with an AirPore™ (Qiagen Inc.) sheet and incubate at 37⁰C with shaking at 325 RPM for approximately 20 hours.

2. Pellet cells by centrifugation for 20’ at 3,200 RPM in a Sorvall RT7 at 4⁰C with RTH-250 rotor, ~1700 xg. Decant and drain pellets. If duplicates are used, transfer culture fluid from the duplicate block to the first block. Spin again, decant and drain.

3. Resuspend each pellet in 300ul of ice cold Qiagen buffer R1 with RNAse A (0.2 mg/ml) and RNAse T1 (100 units/ml). Let rest for 10 min. on ice to relax pellets before gentle vortexing or shaking. [1]

4. Add 300 ul of Qiagen buffer R2 (lysis buffer) per well. Seal wells with plastic sealing tape and mix by gentle inversion 5 times. Incubate at room temperature for 5 minutes (most critical timed step).

5. Add 300 ul of cold Qiagen buffer R3 (neutralization buffer) per well. Seal wells with plastic sealing tape and mix by gentle inversion 5 times. Incubate on ice for 5 minutes.

6. Add 50 ul of ProCipitate™ (LigoChem Inc.) per well. Invert gently several times during a 5 minute room temperature incubation. Let stand at room temperature for 1 minute before transferring to filter plate.

7. Using a wide bore pipette tip, transfer the lysate to a Qiagen TurboFilter™ (Qiagen Inc.) or QiaFilter™ (Qiagen Inc.) plate positioned over a 96 deep-well collection block inside a vacuum manifold. Transfer 600 ul at a time, collecting the top half (with flocculent ppt.) first. Vacuum filter using approximately 300-500 mm Hg until filtration is complete, about 5-20 minutes.

8. Remove lysate filtrate collection block from manifold. Add 2ul of glycogen type II (Sigma Inc.) at 1ug/ul and ice-cold isopropanol (0.7x volume of filtrate) to each well. Seal with plastic sealing tape and mix. Incubate for ~60 minutes at -20⁰C.

9. Centrifuge for 60 minutes at 4?C at 1900 xg (3400 RPM). Decant gently. [2]

10. Wash pellets with 500 ul of 70% ethanol. Spin for 10 minutes at 4⁰C at 1900 xg. Decant block gently and blot dry.

11. Air dry or speed-vac to just dry and resuspend pellets in 1 mM Tris, pH 8.0. Use 10ul of resuspension buffer per ml of starting culture fluid.

【1】Steps 3 through 8 can be carried out using a robotics system such as the Qiagen BioRobot 9600. When doing so, R1, R3, and isopropanol are at ambient temperature and the ProCipitate™ step is excluded.

【2】G-force is limited by the centrifuge available. Centrifuges which allow higher g-force can be used to reduce centrifugation time.