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

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01/21/11

 * &#x2713; Oligos: order sequencing primers for pSB31
 * &#x2713; PCR cloning: p65 and SP1AB activation domains
 * &#x2713; Oligos: order new p65 BioBrick oligos
 * &#x2713; Cultures: pSB31 from rt plate (2); 5 mL each

Oligos > Use sequence around EcoRI cloning site (from Manching) > IDT DNA
 * 1) seq_pSB31 f: 5'-CGTTTAGTGAACCGTCAGATC
 * 2) seq_pSB31 r: 5'-CATTCTAGTTGTGGTTTGTCC

> p65 (RelA), aa's 288-548/ bp 862-1644; Contains 2 PstI sites
 * 1) BB_p65 f1: 5'- CCTTTCTAGA tacctgccagataca
 * 2) BB_p65 r1: 5'- AAGGCTGCAGCGGCCGCTACTAGT ctgactcagcagggc
 * 3) 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 > Primers
 * 1) FTRx no-rot, 7/08/10
 * 2) n.t. kd 8 dy, 11/08/09
 * 1) BB_p65 f/ r
 * 2) BB_SP1A f/ BB_SP1AB r

> 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

--> 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

> Ligations

--> ~10 min./ r.t.; Add to 30 μL DH5α turbo --> Forgot to dephos. the vector! Re-do tomorrow.


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