User:Morgan L. Paull/Notebook/C.Dog V1.0 - T7 Promoter/2011/06/21

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Nanodroped GFP and RFP backbone plasmids

  • Concentrations a little low, in the 15 to 60 μL range.
  • Next round of minipreps I will do only one miniprep tube per culture tube, to increase concentration

Digesting Backbones with BsaI

GFP

  • 50 μL G1.1 miniprep (~1500 ng)
  • 1.5 μL BsaI enzyme
  • 6 μL NEB buffer #4
  • 2.5 μL DH2O

RFP

  • 50 μL G1.1 miniprep (~2500 ng)
  • 1.5 μL BsaI enzyme
  • 6 μL NEB buffer #4
  • 2.5 μL DH2O

Began incubating each in 37°C water bath at 11:45 am, removed at 2:45pm, ran on .7% agarose gel, cut out the lowest band (digested backbone). Performed gel purification protocol, skipping isopropanol step at Colin's advice.


Oligos

Received in mail:

  • 1361 - Makoff1_FW_WT
  • 1362 - T7-promoter-FW
  • 1363 - T7-promoter-RV

Centrifuged tubes at 15,000 rpm for 10 mins, resuspended with (nmol*10) μL of DH2O:

  • 1361 at 26.5 nmol --> 265 μL DH2O
  • 1362 at 26.8 nmol --> 268 μL DH2O
  • 1363 at 33.7 nmol --> 337 μL DH2O

Phosphorylating Oligos

In PCR Tubes

  • 1 μL T4 Polynucleotide Kinase (PNK)
  • 2 μL NEB ligase buffer (10x)
  • 2 μL oligo (oFABs 1362, 1263, 1361, and 980)
  • 15 μL DH2O

In PCR machine

  • 37°C for 60 mins
  • 95°C for 3 mins
  • 4°C indefinitely

Removed the completed reaction tubes at the end of the day, and put them in my box in the -20°C freezer.