User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/22

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01/22/11

 * &#x2713; Re-do ligation: SP1AB w.t./V0120
 * &#x2713; Miniprep: pSB31 (2)
 * &#x2713; Assemblies: KAH160, 165, 170 into pSB31

Re-do ligations from 01/21/11

> Dephosphorylation (Roche)
 * 1) V0120 X/S vector

> Ligations

--> ~1 hr./ r.t.; Add to 30 μL DH5α turbo

Assemblies
 * 1) KAH160/pSB31: KAH169/(E/S blunt)/1147 + pSB31/(E blunt)/5127
 * 2) KAH165/pSB31: KAH165/(E/S blunt)/991 + "
 * 3) KAH170/pSB31: KAH170/(E/S blunt)/955 + "

> Digests (Fermentas FD) --> Digest pSB31 vector w/ EcoRI; 2 rxns --> Do not gel purify

--> Zymo clean EcoRI cut vector; elute w/ 25 μL dH2O

> Blunting (NEB Quick Blunting kit) --> Blunt all DNA (vectors and inserts)

--> Clean vector DNA using Zymo DNA Clean & Concentrator; elute w/ 20 μL dH2O
 * 1) KAH160 (max. vol.) [final] = 2.7 ng/μL
 * 2) KAH165 (max. vol.) [final] = 6.9 ng/μL
 * 3) JDS170 (max. vol.) [final] = 4.6 ng/μL
 * 4) pSB31 (max. vol., measure after cleaning)

> Measure concentration

> Dephosphorylation (Roche) --> pSB31 (E blunt); 3 rxns

> Ligations

--> ~1 hr./ r.t.; Add to 30 μL DH5α turbo


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