User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/27

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01/27/11

 * &#x2713; Assemblies: p65 w.t. (PCR); miR sensors
 * &#x2713; Sequencing order: Pc-TF/pSB31 clones; SP1AB mut clones

Assemblies --> Include set from 01/25/11
 * 1) p65 w.t.: p65 w.t./(X/S PCR)/783 + V0120/(X/S dp)/~3200 &#x2713;
 * 2) KAH179/176fwd/MV8: KAH179/(E/S)/2941 &#x2713; + KAH176/MV8/(E/X)/7785&#x2713;
 * 3) KAH180/176fwd/MV8: KAH180/(E/S)/2743 &#x2713; + "
 * 4) KAH179/177fwd/MV8: KAH179/(E/S)/2941 + KAH177/MV8/(E/X)/7978 &#x2713;
 * 5) KAH180/177fwd/MV8: KAH180/(E/S)/2743 + "
 * 6) KAH179/178fwd/MV8: KAH179/(E/S)/2941 + KAH178/MV8/(E/X)/7989 &#x2713;
 * 7) KAH180/178fwd/MV8: KAH180/(E/S)/2743 + "

> PCR --> p65 from cDNA templates (Note: p65 up-regulated in stressed cells; try cDNA from rotenone-stressed cells)
 * 1) FTRx no rot., 7/08/10
 * 2) FTRx + rot., 7/08/10

--> 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 PCR; elute w/ 25 μL dH2O

> Digests (Fermentas FD)

> Measure conc.'s

> Ligations

--> R.T./ ~15 min.; Add to 50 μL DH5α turbo

Sequencing Order (Genewiz) --> Results: 1/28/11


 * 1) KAH160/pSB31-9, seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
 * 2) KAH160/pSB31-9, seq_pSB31 rev: failed (non-specific)
 * 3) KAH165/pSB31-1 (#7), seq_pSB31 fwd: insert in reverse orientation
 * 4) KAH165/pSB31-1 (#7), seq_pSB31 rev: failed (ns)
 * 5) KAH165/pSB31-4 (#10), seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
 * 6) KAH165/pSB31-4 (#10), seq_pSB31 rev: failed (ns)
 * 7) KAH170/pSB31-3 (#2), seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
 * 8) KAH170/pSB31-3 (#2), seq_pSB31 rev: failed (ns)
 * 9) KAH170/pSB31-5, seq_pSB31 fwd: insert in reverse orientation
 * 10) KAH170/pSB31-5, seq_pSB31 rev: failed (ns)
 * 11) SP1AB 2/3mut-1, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2 (keep this clone)
 * 12) SP1AB 2/3mut-1, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
 * 13) SP1AB 2/3mut-2, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2
 * 14) SP1AB 2/3mut-2, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
 * 15) SP1AB 2/3mut-3, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2
 * 16) SP1AB 2/3mut-3, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
 * 17) SP1AB 2/3mut-4, P0001: n/d
 * 18) SP1AB 2/3mut-4, P0002: n/d
 * 19) SP1AB 2/3mut-5, P0001: n/d
 * 20) SP1AB 2/3mut-5, P0002: n/d
 * 21) SP1AB 2/3mut-6, P0001: n/d
 * 22) SP1AB 2/3mut-6, P0002: n/d

--> Pc-TF/pSB31: Success! Got all three clones for ES cell transfection experiment. Maxi prep and send to Manching. Reverse primer failed (discard/ design new primer). --> SP1AB mutagenesis: U2OS seems to have two missense mutations in the SP1AB activation domain (bp 230: Gln -> Arg; bp 1141: Ser -> Gly). Keep clone #1 for mutation of PstI #1 (design new longer primer).


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