User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/14

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

01/14/10

 * &#x2713; Minipreps: KAH117, 118/MV2 (6 each); KAH94 (2), KAH95 (2) retransformations
 * &#x2713; Assemblies: KAH94, 95/JDS47; KAH94, 95/JDS48
 * &#x2713; Re-transform: KAH117, 118/MV2

Minipreps > Check with E/P digests

--> Re-transform 117, 118/MV2 to isolate clones without mysterious extra bands

Assemblies
 * 1) KAH94/JDS47: (1) KAH94/(N, blunted)/5011 + JDS47/(BglII, blunted)/7509 &#x2713;
 * 2) KAH95/JDS47: (2) KAH94/(N, blunted)/5083  + "
 * 3) KAH94/JDS48: KAH94/(N, blunted)/5011 + JDS48/(BglII, blunted)/7519 &#x2713;
 * 4) KAH95/JDS48: KAH94/(N, blunted)/5083  + "

> Digests (Fermentas FD)

> Measure conc.'s of inserts

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


 * 1) KAH94 (max. vol.) [final] = 3.3 ng/μL
 * 2) KAH95 (max. vol.) [final] = 2.8 ng/μL
 * 3) JDS47 (use max. vol, measure after cleaning)
 * 4) JDS48 (use max. vol, measure after cleaning)

> Clean vector DNA using Zymo DNA Clean & Concentrator

> Dephosphorylation (Roche)

> Ligations


 * }