User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/14

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01/14/11

 * &#x2713; Assemblies: KAH173, 174
 * &#x2713; Order primers: for p65 and SP1 activation domain BioBricks

Assemblies > Re-do KAH173 using dephos vector and max. insert
 * 1) KAH173: HPK/(E/S)/516 &#x2713; + KAH73/(E/X)/~5638 &#x2713;
 * 2) KAH174: KAH75/(E/S)/558 &#x2713; + (1) KAH57/(E/X)/~4626

> Digests (Fermentas FD)

> Measure conc.'s

> Dephosphorylation (Roche)
 * 1) KAH73(E/X)

> Ligations (1/15/11)

--> 10 min./ R.T.; add to 30 μL DH5α turbo

New BioBricks: p65, SP1 activation domains --> Will PCR-clone these from human genomic DNA

> p65 --> Note (1/21/11): wrong ref sequence; redesign primers!
 * a.k.a. NFκB p65 subunit, RELA
 * 261 amino acids from C-terminus (excluding stop codon) (Liu et al., 2001)
 * Contains two adjacent PstI sites
 * Primers:
 * 1) BB_p65 f1: 5'- CCTTTCTAGA GGAGCTGATCTGACT
 * 2) BB_p65 r1: 5'- AAGGCTGCAGCGGCCGCTACTAGT GATACAGACGATCGT
 * 3) mut_p65 1: 5'-ATCAAACTGCAaCTGCAaCAGGGCCTCTGA

> SP1
 * Has two activation domains that are sufficient for transcription at TATA/Inr promoters
 * SP1AB is unable to activate a TATA-only promoter; will be interesting to see how this impacts global expression
 * SP1A: aa's 83-262, 180 aa, 540 bp; 1 PstI site
 * SP1B: aa's 263-542, 280 aa, 840 bp; 2 PstI sites
 * SP1AB: aa's 83-621, 539 aa, 1617 bp; 3 PstI sites (clone & mutate this first, then use as template for A and B)
 * Primers
 * 1) BB_SP1A f1: 5'- CCTTTCTAGA GGCCCGAGTCAGTCA
 * 2) BB_SP1A r1: 5'- AAGGCTGCAGCGGCCGCTACTAGT CCCATTCAGGGCCAC
 * 3) mut_SP1A 1: 5'-AACAGCTGCAaTTTGCTGCC
 * 4) BB_SP1B f1: 5'- CCTTTCTAGA AACATCACCTTGCTA
 * 5) BB_SP1B r1: 5'- AAGGCTGCAGCGGCCGCTACTAGT GATTCCTGAAGTACC
 * 6) mut_SP1B 1: 5'-CGTTTCTGCAaCTACCTTGA
 * 7) mut_SP1B 2: 5'-TACAGCTGCAaAACCTCCAA
 * 8) BB_SP1AB r1: 5'- AAGGCTGCAGCGGCCGCTACTAGT GCCAGGATCCCCCGA


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