User:Lu Wang/Notebook/Team Allergy/2010/06/29

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Procedures

Today, we did not do work with amiRNA and focused our efforts on hpRNA. We obtained undigested V0120 with a death gene insert from Team Flavor, and we have inserts from previous PCR reactions that all need to be digested and ligated into V0120.

Procedures:

  1. Digestion of Allergen Panel and V0120 with Xba and Pst
  2. Ligation of Allergen inserts into Vector Backbone
  3. Transformation of E. Coli

Results

Digestions

Digestion Reactions
Bet GFP LTP LTP(.5) GFP(.5) Backbone (V0120 w/o a death gene)
DNA 28 28 28 28 28 5
FD Buffer (10x) 4 4 4 4 4 2
diH2O 6 6 6 6 6 11
EcoRI 1 1 1 1 1 1
SpeI 1 1 1 1 1 1


Concentrations:

Bet: 58.2 ng/uL; LTP (.5): 38.6 ng/uL; GFP (.5): 43.9 ng/uL; GFP: 29.1 ng/uL; LTP: 60.4 ng/uL; Backbone: 20.2 ng/uL


Ligations for full amiRNA and V0120 (w/o death gene)

note: .5 denotes multistep pcr reaction from yesterday in which .5 uL of part 3 of gfp/ltp were use in one of the giant assemblies of parts 1,2,3 w/ primers A&B

Ligation Reactions
Bet GFP LTP LTP(.5) GFP(.5)
DNA Insert 1 1 1 1 1
T4 DNA ligase buffer 10 10 10 10 10
diH2O 7 7 7 7 7
T4 DNA ligase 1 1 1 1 1
DNA Backbone 1 1 1 1 1


Transformation ( see transformation protocol)

Transformed amiRNA into turbo cells and plated on LB amp plates--will check for colonies tommorow (Plated at ~ 4pm)