Transgene Construct Preparation Protocol

Transgene Preparative Restriction Digest – 2 enzymes

Digest: 40 ul total volume (do 6 digests)

_______ ul DNA prep______(~3ug)
_______ ul H20
4 ul 10X buffer _________
4 ul 10X BSA
1.5 ul of each enzyme__________

If using 1 enzyme, use 3ul of enzyme per restriction digest

Incubate minimum 4 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 5 minutes after pouring should not set-up more than 7 - 10 minutes before adding cut gel bands
Place gel bands into new gel and let stand RT until completely set up (~ 1 hour)
Run overnight 30V (= 2nd 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 5 minutes after pouring should not set-up more than 7 - 10 minutes before adding cut gel bands
Place gel bands into new gel and let stand RT until completely set up (~ 1 hour)
Run overnight 30V (= 3rd 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 2 photos – 1 for your records and 1 for the MGC
*NOTE: For BAC Transgenes please contact Mia (mia@wustl.edu) for the BAC preparation protocol.

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