User:Morgan L. Paull/Notebook/C.Dog V1.0 - T7 Promoter/2011/06/22

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] C.Dog T7 Promoter Project
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Nanodropped Gel Purified Backbone from yesterday

 * GFP at 3.1 ng/μL and 4.51 260/280
 * RFP at 5.4 ng/μL and 3.74 260/280

Annealing Oligos

 * oFAB 1362 + oFAB 1363 --> T7 Promoter
 * oFAB 1361 + oFAB 980 --> Makoff RBS 1

In PCR tubes

 * 5 μL Phosphorylated FW oligo (from yesterday)
 * 5 μL Phosphorylated RV oligo (from yesterday)
 * 90 μL DH2O

In PCR machine

 * 95°C for 3 mins
 * removed from machine, placed on benchtop for 40 minutes, from 11:05am until 11:45am

Nanodroped Annealed Oligos

 * T7 Promoter: 59.5 ng/μL and 4.22 260/280
 * Makoff RBS 1: 64.3 ng/μL and 3.33 260/280

Insert Ligation Reactions (one GFP, one RFP)

 * 1  μL (GFP or RFP) vector backbone, BsaI digested and purified
 * 2  μL T7 promoter annealed oligos, phosphorylation (diluted 1 in 10 to 5.95 ng/μL)
 * 2  μL Makoff 1 RBS annealed oligos, phosphorylation (diluted 1 in 10 to 6.43 ng/μL)
 * 1  μL NEB T4 ligase buffer (10x)
 * 0.5 μL NEB T4 ligase
 * 1.5 μL DH2O

Backbone Controls (one GFP, one RFP)

 * 1  μL (GFP or RFP) vector backbone, BsaI digested and purified
 * 7.5 μL DH2O

Total of 4 reactions, left on benchtop to ligate for 35 mins, from 12:30pm to 1:05pm.

Transformations

 * added 25 μL of competent cells, thawed on ice
 * let sit on ice for an hour, from 1:15 to 2:15pm (I could have done only 30 mins)
 * heat shocked at 42°C for 30 secs
 * put on ice briefly, added 100 μL SOC
 * put in 37°C shaker for 40 mins, from 2:25 until 3:05
 * streaked on Kan 50 plates
 * put in 37°C still incubator, by the windows 1 bench south of the BIOFAB bench.


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