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Preparation of BAC DNA for Injection

Day 1
BAC containing bugs were grown in 250 mls of LB overnight.

Day 2
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 yielded ~ 30 ug of BAC DNA as measured by a spec.

Day 3
For constructs subcloned into BAC-Link vectors, use 10 ug of BAC and cut it with an I-PPO restriction enzyme (promega). Total volume of digest reaction should be ~ 200 to 250 ul.

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

Equilibrate the column with injection buffer (10mM Tris pH7.5, 0.1mM EDTA, 100mM NaCl) ~ ten column volumes. Column flow rate is ~ 1ml/three minutes so it will take 30 minutes to equilibrate the column.

50 mls of injection buffer:

  • 500 ul of 1M Tris pH 7.5
  • 10 ul of 0.5 M EDTA pH 8.0
  • 1 ml of 5 M NaCl
  • 48.5 ml of water

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

Digest for two to three 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 2 ug/ml (6ul of 100 ug/ml lamda DNA into 294ul TE). Then make three more consecutive 1/10 dilutions to generate the following standards, 2 ug/ml, 0.2 ug/ml, .02 ug/ml, .002 ug/ml) (dilutions should be 30 ul into 270 ul of TE).
  • Dilute Picogreen fluorescent substrate (20 ul into 4 ml of TE).
  • Dilute your unknown fractions (20 ul into 180 ul TE).
  • Pipet 100 ul of standards and unknowns into 96well plate in duplicate.
  • Pipet 100 ul of substrate into each well.
  • Measure on plate reader (Fluorocount).

Using linear regression, calculate concentrations of unknowns.

Concentration
RFU
 
AVE
DEV
Blank Subtracted
0 318 314 316 2.828427 0
1 421 377 399 31.1127 83
10 1028 983 1005.5 31.81981 689.5
100 6560 6792 6676 164.0488 6360
1000 56256 57600 56928 950.3515 56612

Unknowns
   
AVE
DEV