User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/21

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01/21/11 ✓ Oligos: order sequencing primers for pSB31 ✓ PCR cloning: p65 and SP1AB activation domains ✓ Oligos: order new p65 BioBrick oligos ✓ Cultures: pSB31 from rt plate (2); 5 mL each Oligos > Use sequence around EcoRI cloning site (from Manching) > IDT DNA seq_pSB31 f: 5'-CGTTTAGTGAACCGTCAGATC seq_pSB31 r: 5'-CATTCTAGTTGTGGTTTGTCC > p65 (RelA), aa's 288-548/ bp 862-1644; Contains 2 PstI sites BB_p65 f1: 5'-CCTTTCTAGAtacctgccagataca BB_p65 r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTctgactcagcagggc mut_p65 1: 5'-ggccctgctgcaactgcaatttgatgatga PCR > First, PCR and clone p65 and SP1AB into V0120 using E/S (w.t. parts contain PstI sites) > Use U2OS cDNA as template FTRx no-rot, 7/08/10 n.t. kd 8 dy, 11/08/09 > Primers BB_p65 f/ r BB_SP1A f/ BB_SP1AB r Reagent Volume cDNA 1.0 10 μM primers 1.0 2x GoTaq mix 25.0 dH2O 23.0   50.0 μL > BioRad DNA Engine, block A 95 °C/ 3 min. [95°C/ 30 sec.; 55°C/ 30 sec.; 72°C/ 1 min.] x30 72°C/ 3 min. 4°C/ ∞ > Zymo clean; elute w/ 25 μL dH2O > Digests (Fermentas FD) --> X/S digest Reagent Volume   Expected: 1. FTRx/ p65 = 783 2. nt kd/ p65 = 783 3. FTRx/SP1AB = 1617 3. nt kd/SP1AB = 1617 30 μL/lane, 1% agarose PCR 25.0 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. --> p65 did not work --> Note: Double checked p65 sequence. Ref sequence was wrong. Discard old primers and replace with new primers designed from correct human p65 CDS. --> Proceed w/ SP1AB cloning > Measure conc.'s Sample OD260 260/280 ng/μL 2. SP1AB w.t. (X/S) 0.177 2.02 8.9 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. SP1AB w.t.(X/S)/1617, 25 ng + V0120(X/S)/~3200, 25 ng SP1AB w.t./V0120 1:1 2. V0120(X/S)/ 25 ng     1 2 Insert DNA 3.0 --- Vector DNA 1.0 1.0 2x lgn buf (Roche) 5.0 ### T4 ligase (NEB) 1.0 1.0 dH2O --- 3.0   10 μL 10 μL --> ~10 min./ r.t.; Add to 30 μL DH5α turbo --> Forgot to dephos. the vector! Re-do tomorrow.