Transgene Construct Preparation Protocol

Transgene preparative restriction digest (two enzymes)

Digest: 40 ul total volume (do six digests)

_______ ul DNA prep ______ (~3ug)
_______ ul H²0
4 ul 10X buffer _________
4 ul 10X BSA
1.5 ul of each enzyme __________

If using one enzyme, use 3ul of enzyme per restriction digest

Incubate minimum four hours, 37C (note if different temp required – check enzyme list)
Run on 1% low melt gel (100ml) overnight, 30V, 20ul of ladder
Let run all day the next day at 30V – DO NOT turn up the voltage
Cut gel bands and place in microtubes – hold at 4C until next gel is ready
Pour 1% low melt midi-gel (100ml) without comb, check the gel five minutes after pouring should not set-up more than seven to ten minutes before adding cut gel bands
Place gel bands into new gel and let stand RT until completely set up (about one hour)
Run overnight 30V (= second overnight)
Let run all day the next day at 30V – DO NOT turn up the voltage
Cut gel bands and place in microtubes – hold at 4C until next gel is ready
Pour 1% low melt midi-gel (100ml) without comb, check the gel five minutes after pouring should not set-up more than seven to ten minutes before adding cut gel bands
Place gel bands into new gel and let stand RT until completely set up (about one hour)
Run overnight 30V (= third overnight)
Let run all day the next day at 30V – DO NOT turn up the voltage
Cut gel bands, store 4C or proceed with Qiaex II purification
Qiaex II purify – ELUTE with transgene injection buffer

Transgene Injection Buffer

Tris (pH 7.4): 10mM
EDTA: 0.1mM
________
OD260/280: concentration _____________ ratio: ______________
Run 100ng on 1% gel to check – take two photos – one for your records and one for the MGC

*NOTE: For BAC Transgenes please contact Mia (mia@wustl.edu) for the BAC preparation protocol.