Section on Neural Gene Expression

Before Beginning:

Prepare a 20 h culture of BAC containing bacteria in 2X YT and appropriate antibiotic. The OD₆₀₀ of the final culture should be 5.0 ± 0.5.

Add RNAse A to E1 to a final concentration of 400 µg/mL.

Increase NaCl concentration in Wash Buffer (E5) from 0.8 M to 0.9 M NaCl by adding 0.58 g NaCl per 100 mL E5. Conductivity should be 72 mS. This increase in salt will reduce the RNA contamination in the BAC prep.

Pre-warm Buffer E6 to 50^oC.

NOTE: In this application, we recommend 400 µg RNAse A/mL of Suspension Buffer (E1) because of the extremely low copy number of BACs.

1. Equilibrate the column with Equilibration Buffer (Buffer E4) Allow the solution in the column to drain by gravity flow.

	Miniprep	Midiprep	Maxiprep
Volume of E4	2 ml	10 ml	30 ml

Pellet the cultured cells by centrifugation at 9000 x g for 15 min.  THOROUGHLY remove all media after pelleting and before resuspending.

	Miniprep	Midiprep	Maxiprep
Volume of BAC culture	10-25 ml	100 ml	200 ml

Resuspend the cells in Cell Suspension Buffer (Buffer E1) containing 400 µg/mL RNase A.

	Miniprep	Midiprep	Maxiprep
Volume of E1	2 ml	8 ml	40 ml

Lyse the cells with Cell Lysis Solution (Buffer E2.) Mix IMMEDIATELY. Mix gently but thoroughly until a homogenous mixture is obtained. Due to the release of genomic DNA, the mixture is very viscous at this stage. Incubate at room temperature for 5 min.

	Miniprep	Midiprep	Maxiprep
Volume of E2	2 ml	8 ml	40 ml

Neutralize the lysis mixture by adding Neutralization Buffer (Buffer E3).  Mix IMMEDIATELY. Mix thoroughly, but do not vortex. Vortexing can result in shearing of the DNA.

	Miniprep	Midiprep	Maxiprep
Volume of E3	2 ml	8 ml	40 ml

Centrifuge the lysate for 10 min >15,000 x g at room temperature. Collect the supernatant into a fresh tube. Collection of the supernatant should be performed with a pipet, not by decanting.

Apply the cleared lysate supernatant to the column. Allow the lysate to run through the column by gravity flow. DO NOT force out the remaining buffer.

Wash the column once with Wash Buffer (Buffer E5). Allow the wash to flow through the column by gravity. Discard the flow-through.

	Miniprep	Midiprep	Maxiprep
Volume of E5	2.5 ml	10 ml	60 ml

Elute the DNA from the column into a clean tube by adding Elution Buffer (E6) that has been warmed to 50¡C. Allow the solution to drain by gravity flow. Do not force out remaining solution. Prewarming of E6 will enhance the release of high-molecular weight DNA.

	Miniprep	Midiprep	Maxiprep
Volume of E6	0.9 ml	5 ml	15 ml

Precipitate the DNA by adding isopropanol (0.7 volumes) to the eluate. Mix and centrifuge DNA for 30 min at >12,000 x g at 4^oC. Carefully discard the supernatant.

	Miniprep	Midiprep	Maxiprep
Volume of isopropanol	0.63 ml	3.5 ml	10.5 ml

Wash the BAC DNA pellet with 80% ethanol and centrifuge at >15,000 x g at 4^oC for 5 min. Carefully and fully pipet off the ethanol wash. Air dry the pellet for 10 min.  Dissolve the DNA in 10 mM Tris pH 8.0.

	Miniprep	Midiprep	Maxiprep
Volume of Tris	10 µl	50-100 µl	200-400 µl

We have achieved yields of a ~100 kb BAC molecule of approximately 40 µg DNA / 100 ml culture with the above procedure.

¹Currently available from Life Technologies products of Invitrogen.