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
Ligating T7 + Makoff 1 + Makoff 2 + backbone
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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C.Dog T7 Promoter Project
