User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/07

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07/07/11

 * &#x2713; Minipreps: mCherry, 5xGal4 retransformations
 * &#x2713; Assemblies: KAH204, 205, 206, 207, 208, 209
 * &#x2713; Site directed mutagenesis: p65 (new primers)
 * &#x2713; Digest: investigate weird miR-luc sensor constructs; check KAH196, 177, 198/MV8

Minipreps > Check with E/P digests

Assemblies
 * 1) KAH204: (1) mCh/(S/P)/705 + (2) SP1AB/(X/P)/1617
 * 2) KAH205: " + (3) SP1A/(X/P)/540
 * 3) KAH206: " + (4) SP1B/(X/P)/840
 * 4) KAH207: (5) KAH203/(S/P)/155 + (8) KAH182/(X/P)/1675
 * 5) KAH208: (6) KAH201/(S/P)/203 + "
 * 6) KAH209: (7) KAH202/(S/P)/251 + "

> Digests (Fermentas FD)

> Measure conc.'s

> Ligations

Site-directed Mutagenesis > Stratagene Quick Change mutagenesis kit. --> Try to mutate second PstI site with forward and reverse primers...
 * 1) p65 (~4 kb): tried to mutate w.t. clone (two PstI sites within single primer); last attempt mutated only the first site (3/25/11)
 * 1) Primer mut_p65 2 (plus strand)
 * 2) Primer mut_p65 2 (minus strand)

--> 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/ ∞

(7/08/11) > DpnI Digest (gets rid of methylated template DNA)
 * 1) p65 + strand mutation
 * 2) p65 - strand mutation
 * 3) 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

07/09/11 --> Results
 * p65 +strand mut: 0 colonies (0 on neg ctrl.)
 * p65 -strand mut: 0 colonies (0 on neg ctrl.)

Minipreps > Check with ApaLI > Refer to 6/04/11 for correct 196, 198 bands (E/ApaLI)

--> Everything looks correct. Use these minipreps for re-cloning final miR-luc sensor set. --> Note: pre-cut vector KAH177/MV8 worked for 183-177/MV8 (see 6/07/11); continue using this vector) --> Re-cut 196 and 198/MV8, throw out old cut vectors


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