Protocols

Preparation of BAC DNA for injection.

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Day1
BAC containing bugs were grown in 250mls of LB overnight. 

Day2
BAC DNA was prepared using Qiagen’s large construct kit.  This kit includes an exonuclease digestion step that removes sheared BAC DNA and Bacterial DNA.  The last prep I did yielded ~30ug of BAC DNA as measured by a spec. 

Day3
For constructs subcloned into BAC-Link vectors, use 10ug of BAC and cut it with an I-PPO restriction enzyme (promega).  Total volume of digest reaction should be ~200-250ul. 

During digest, set up a 2-2.5ml sepharose CL-4B column (Sigma CL4B200-100ml).  I use Biorad poly-prep chromatography columns to pour the sepharose. 

Equilibrate the column with injection buffer (10mM Tris pH7.5, 0.1mM EDTA, 100mM NaCl) ~10 column volumes.  Column flow rate is ~1ml/3minutes so it will take 30 minutes to equilibrate the column.
           
50mls of injection Buffer:           500ul of 1M Tris pH 7.5
                                                            10ul of 0.5M EDTA pH 8.0
                                                            1ml of 5M NaCl
                                                            48.5ml of water

Prepare 12 tubes for collection.  Collect ~250-300ul per fraction. 

Digest for 2-3 hours then run digest through column.

Immediately start collecting fractions after sample is applied.  After sample enters resin, gradually add injection buffer to column.

Measure DNA concentration using Fluorescent DNA assay.
Using PicoGreen, carryout a DNA content assay on all the fractions.  Run Fractions on pulse field gel to verify optimal fraction.

In preparation for assay:

Dilute DNA standard to 2ug/ml (6ul of 100ug/ml lamda DNA into 294ul TE).  Then make 3 more consecutive 1/10 dilutions to generate the following standards, 2ug/ml, 0.2ug/ml, .02ug/ml, .002ug/ml) (dilutions should be 30ul into 270ul of TE)

Dilute Picogreen fluorescent substrate (20ul into 4ml of TE)
Dilute your unknown fractions (20ul into 180ul TE)

1. Pipet 100ul of standards and unknowns into 96well plate in duplicate
2. Pipet 100ul of substrate into each well
3. Measure on plate reader (Fluorocount)

 
Using linear regression, calculate concentrations of unknowns.