User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/28

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01/28/10

 * &#x2713; Assembly: KAH139/V0201
 * &#x2713; PCR SOEing: EF1α promoter (mutate two PstI sites)

PCR SOEing > EF-1α promoter (for miR sensor optimization); has 2 PstI sites > Step 1: mutate one of the PstI sites (later, use the mutated BioBrick for mutation of other PstI) --> *Done in case PCR for mutating 1st PstI site fails
 * 1) Fwd EF1A / EF-1a Pst1 r, amplicon 1 (1st PstI site)
 * 2) EF-1a Pst1 f / Rev EF1A, amplicon 2 (1st PstI site)
 * 3) Fwd EF1A / EF-1a Pst2 r, amplicon 1b (2nd PstI site)*
 * 4) EF-1a Pst2 f / Rev EF1A, amplicon 2b (2nd PstI site)*

> PCR round 1

--> BioRad Block A
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞

--> Clean bands 1 and 4 using Zymo clean & concentrator; elute w/ 20 μL dH2O

> Alternative Step 2 --> 1/2 fragment PCR didn't work, but mutation PCR for each end worked. Try using these as megaprimers (with EF-1α template) to create both PstI mutations at the same time. --> Small population of amplicons should contain both mutations. Digestion w/ PstI should cut all products except for double-mutant fragments.

--> BioRad Block A
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞

--> Very faint product appears to be correct size. Repeat this PCR with 4 rxn's and 2.0 μL each megaprimer (tomorrow)

Assemblies
 * 1) KAH139/V0201: &#x2713; KAH139/(N/S)/2945 + &#x2713; V0201/(N/S)/5157

> Ligations


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