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

From OpenWetWare

Jump to: navigation, search
C.Dog T7 Promoter Project Main project page
Previous entry      Next entry

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.




Personal tools