IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/05

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Colonies from yesterday's ligations




Gel extraction of EcR-Gal Bam Nco

 * applied 3x gel volume of QG buffer (in μL) to each respective tube
 * placed in 42°C bath until the gel was melted
 * added 1 gel volume of isopropanol to each, and vortexed
 * applied contents of each tube to a QIAquick spin collumn
 * spun for 1 min
 * added 500μL QG buffer (to dissolve any remaining agarose)
 * spun for 1 min
 * added 750μL PE buffer
 * spun for 1 min, discared flow-through
 * spun for another minute
 * placed QIAquick collumn in a fresh, labeled eppendorf tube, applied 50μL EB buffer to elute, and spun for 1 min

Digestions
VP16
 * 1μL xba
 * 1μL Pst
 * 5μL VP16(2) miniprep
 * 1μL 10x FD green buffer
 * 2μL DH2O

5xGalUAS
 * 1μL xba
 * 1μL Pst
 * 2μL 5xGal4UAS (from Karmella)
 * 1μL 10x FD green buffer
 * 5μL DH2O

PhyB
 * 1μL Bam
 * 1μL Nco
 * 16μL PhyB
 * 2μL 10x FD green buffer

Sending EcR-Gal for sequencing
Sealed tubes with parafilm. Submited Gal-EcR 1, 7, 10, and 12 (the one's with inserts on the digestion gel from 8/4)
 * 5μL of each primer, either fwd or rev, at 1/20 dilution
 * 10μL of sample and DH2O, such that sample is at 500ng total mass

Transformation of J45004

 * 5μL of registry DNA was transformed with 15μL TURBO cells; mixture was plated on LB+AMP and left to grow O/N

Team Allergy
''Concentrations: V9 (9.4 ng/uL; V10 (16.4 ng/uL)
 * Gel extracte V9/V10 backbone
 * Ligated ihpRNA inserts into V9/V10 and transformed
 * For our ligations we only used ~ 2uL of backbone (around 18 and 32 ng of backbone)and used a 3x excess of insert
 * Verified that amiRNA stitching of Bet, LTP yielded the proper insert with a low level of background through PCR:
 * Digested Bet,LTP inserts with X+P, B21 with X+P+phosphatase
 * Ligated, transformed
 * 0-7: Bet, corresponding to 65..55 degrees C for stitching Tm (even spacing)
 * 8-15: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing


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