Purdue/2 September 2008

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Restriction Digest of SOS and LacZ

1. Mixed digestion solution:
 * 50uL DNA
 * 7uL NEB2
 * 10.5uL ddH2O
 * 0.5uL BSA
 * 1uL of each enzyme
 * SOS: EcoRI + SpeI
 * LacZ: EcoRI + XbaI
 * This makes SOS the small part cut out (192 bp) and put into the LacZ plasmid (~5000 bp...=LacZ + most of plasmid)

2. Put mixtures at 37C for 1 hour, 25 min.

3. Run through Agarose gel w/ DNA ladder, DNA loading dye (Bio-RAD)
 * Mixed 7uL of loading dye w/ each 70uL tube
 * 10uL ladder in lane 1
 * Lanes 2-4: 20uL LacZ in each lane
 * Lanes 5-7: SOS
 * 120V, 45 min.

4. Overnight stain w/ Fast Blast
 * 5x
 * RT
 * dark
 * rocking

Edited by Janie Stine