Sizing inserts by Pulsed Field Gel Electrophoresis
Follow BAC (and Fosmid) mini-prep protocol III to prepare BAC DNA
Not I digest (per sample : all reagents from New England Biolabs)
| 2.0 ul | NEB buffer #3 | cat # B7003S |
| 0.2 ul | BSA | cat # B9001S |
| 0.5 ul | Not 1 | cat # R0189L |
| 7.3 ul | H20 | |
Make master mix for appropriate number of samples and add 10 ul of this mix to 10ul DNA (or few 100 ng).
Let sit at 37C for at least 2 hours (no limit as to length of time let digest)
Make 1% pulse field gel using 1 g of Seakem LE agarose (Cambrex cat# 50004) and 100 ml of 0.5X TBE
(100 ml 10X TBE plus 1900 ml of water; use rest to run gel). While gel is setting, drain old buffer
(turn off chiller and pump; remove clip on drain tube). Add 2 l of distilled water. Circulate for 5
minutes and drain. Add rest of 2 l of 0.5X TBE and turn on pump and chiller to prechill to 14 C.
When gel is set, remove comb and then remove gel from casting stand. Load size standard (low range pfg, New
England Biolabs cat #N0350S: use razor blade to cut off 1 mm slices and place them in lanes at both edges). Place
gel (on black plate) so it seats within the black collar in the buffer chamber of pulse field gel.
Remove samples from 37C, add 5 ul loading dye (Orange G works), and using a pipette tip with the end cut off,
load 20ul on gel.
Run 10 hours, 14oC, 5.5 V/cm, 5s initial switch time, 20s final switch time
When done, stain with ethidium bromide (6ul/enough water to cover gel)
Should be able to clearly see vector and insert bands.
3/04 CK