User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/05

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07/05/11

 * &#x2713; Cultures/minipreps: KAH201, 202, 203 (3 each)
 * &#x2713; Sequencing order: KAH201, 202, 203
 * &#x2713; Retransform: mCherry E3000
 * &#x2713; (Re-) Assembly: miR sensor 183-177/MV8 (as a control for Daniel's set)
 * &#x2713; Order oligos: new mutagenesis primers for p65

Minipreps > Check with E/P digests

Sequencing order --> Send all samples for Genewiz sequencing --> Use P0001 (fwd) primer for all samples

Note: B433 spacer is actually 42 bp (2 repeat segments, not three)
 * 1) KAH201-1: good (keep this clone)
 * 2) KAH201-2: good
 * 3) KAH201-3: good
 * 4) KAH202-1: good (keep this clone)
 * 5) KAH202-2: good
 * 6) KAH202-3: good
 * 7) KAH203-1: no priming
 * 8) KAH203-2: good (keep this clone)
 * 9) KAH203-3: good

Assembly --> Not sure about previous miR sensor construct vectors; start re-building and check with ApaLI instead of E/ApaLI --> Note: Use KAH177/MV8 E/X digest from 1/25/11, not 5/25/11
 * 1) KAH183-177/MV8: KAH183/(E/S)/2383 &#x2713; + KAH177/MV8/(E/X)/3384 &#x2713;

> Ligations

--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp

Order oligos --> Previous attempts to mutate the second PstI site failed. Order new primers and try both strands...
 * 1) p65 mut 2 (+ strand): TGCTGCAACTGCAaTTTGATGATGAAG
 * 2) p65 mut 3 (- strand): CTTCATCATCAAAtTGCAGTTGCAGCA


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