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	Calculate Concentration of Unknowns
1	356	328	342	19.79899	2.446066
2	337	317	327	14.14214	2.180382
3	5258	5171	5214.5	61.51829	88.74916
4	11509	11249	11379	183.8478	197.9365
5	12998	12997	12997.5	0.707107	226.6038
6	7355	7590	7472.5	166.1701	128.7435
7	2431	2457	2444	18.38478	39.67728