User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/13

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07/13/10

 * &#x2713; PCR: flyeGFP, flyCitrine, flyCFP
 * &#x2713; Assemblies: dsOligo flyNLS, PCR flyeGFP, flymCh, flyCitrine, flyCFP into V0120
 * &#x2713; Transformation: transform DH5α Turbo with plasmids for

PCR
 * 1) flyeGFP (714 bp)
 * 2) flyCFP (711 bp)
 * 3) flyCitrine (711 bp)

BioRad PCR Block A
 * 95°C/ 3 min.
 * [95°C/ 30 sec.; 57°C/ 10 min.; 72°C/ 10 min.] x 30
 * 72°C/ 3 min.
 * 4°C/ ∞

> Zymo clean & concentrate --> Elute w/ 25 μL dH2O > Proceed to digestion

Assemblies
 * 1) KHF01/V0120: &#x2713; flyNLS/(X/P dsOligo)/21 + (1) V0120/(X/P)/~3200
 * 2) KHF02/V0120: (2) flyeGFP/(X/P PCR)/714 + "
 * 3) KHF03/V0120: (3) flyCFP/(X/P PCR)/711 + "
 * 4) KHF04/V0120: (4) flyCitrine/(X/P PCR)/711 + "

> Digests (Fermentas FD)

> Measure conc.'s

> Ligations

--> R.T./ 10 min.; Add 40 μL DH5α Turbo

--> Results: colonies suspiciously low. Not sure if I mistook EcoRI for XbaI tube when cutting V0120. Do over.

Plasmid Transformation > Plasmids from the DePace lab > Transform 0.5 μL (in 10 μL dH2O) into 30 μL DH5α Turbo; plat on 100 μg/mL Amp
 * 1) pJ10:G04380 (flyeGFP+NLS)
 * 2) pJ10:G04376 (flyCFP+NLS)
 * 3) pJ10:G04382 (flyCitrine+NLS)
 * 4) pJ10:G04381 (flymCherry+NLS)
 * 5) pAct5C
 * 6) dH2O (neg ctrl)


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