User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/29

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06/29/11

 * &#x2713; Biobrick PCR: SP1A and SP1B (from s.d. mutated SP1AB)
 * &#x2713; Assemblies: KAH199 (spacer + TK), KAH200
 * &#x2713; SD mutagenesis: re-do mutagenesis of PstI site #2 in p65
 * &#x2713; Cultures/ minipreps: KAH187-198/MV8 (2); concentration of stock is low

Biobrick PCR: SP1A, SP1B > Note: Last step, complete SP1AB mutation, was confirmed on 3/25/11
 * 1) KAH018: (1) SP1A/(X/P PCR)/540 + V0120/(X/P)/~3200 &#x2713;
 * 2) KAH019: (2) SP1B/(X/P PCR)/840 + "

> PCR --> Template: SP1AB Biobrick --> Primers 1: BB_SP1A f/ BB_SP1A r --> Primers 2: BB_SP1B f/ BB_SP1B 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 PCR; elute w/ 25 μL dH2O

> Digests (Fermentas FD)

> Measure conc.'s

> Ligations

--> R.T./ ~15 min.; Add to 30 μL DH5α turbo

Assemblies --> Note: DPRE has PstI sites
 * 1) KAH199: (1) Spacer B433/(E/S dp)/65 + (2) HSVtk TATA DDB81/(E/X)/56
 * 2) KAH200: DPRE KAH014/(E/S)/1740 &#x2713; + 5xGal4 KMC01/(E/X)/93 &#x2713;

> Digests (Fermentas FD) --> Specific notes

> Zymo clean --> Spacer B433 (E/S) insert --> Elute w/ 25 μL dH2O

> Measure conc.'s

--> Dilute 1:2 to get ~42 μg/μL

> Dephosphorylation (Roche) --> Phosphatase-treat the Spacer (E/S) insert

> Ligations --> Use max insert volume for ligation #1 (insert includes old vector)

--> Add 30 μL DH5α Turbo; plate on 100 μg/mL Amp

Site-directed Mutagenesis > Stratagene Quick Change mutagenesis kit. --> Primer mut_p65 1
 * 1) p65 (~4 kb): mutate w.t. clone (two PstI sites within single primer); last attempt mutated only the first site (3/25/11)

--> BioRad PCR (Block A)
 * 95°C/ 1 min.
 * [95°C/ 1 min., 55°C/ 1 min., 65°C/ 8 min (2 min./kb)] x30
 * 65°C/ 1 min.
 * 4°C/ ∞

(6/30/11) > DpnI Digest (gets rid of methylated template DNA)
 * 1) p65 mutation
 * 2) p65 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)

--> Add 1 μL DpnI enzyme to each sample --> 37°C/ 1 hr. --> Transform 30 μL z-DH5α, use 10 μL each sample; Amp plates

07/01/11 --> Results
 * p65: 2 colonies (1 on neg ctrl.)


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