IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/29: Difference between revisions

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*The ligation of Miraculin was done at a 3:1 ratio (insert to vector) due to its larger size (~700 bp)
 
*The ligation of Brazzein was done at a 2:1 ratio due to its smaller size (~200 bp)
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*The ligation of Miraculin was done at a 3:1 ratio (insert to vector) due to its larger size (~700 bp)
*The ligation of Brazzein was done at a 2:1 ratio due to its smaller size (~200 bp)




Revision as of 13:23, 29 June 2010

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Team Allergy

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) Backbone (V0120 w/o a death gene)
DNA 1 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


Transformation ( see transformation protocol)

Transformed amiRNA into turbo cells and plated on LB amp plates--will check for colonies tommorow

Team Fence

B21 plasmid maxiprep

colony inoculated overnight and medium was centrifuged to obtain a pellet.

followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition)

Team Flavor

Results of ligation and transformation of The Big Three (pENTCUP2, NosT, NosT + stop) with B15 backbone into E.coli

A few colonies were found on the pENTCUP2 + Backbone plate, NosT + stop and backbone plate, and b15 backbone control plate. Colonies were picked from the plates and placed in 3ml of LB and agarose in a culture tube. The tubes were placed in the 37°C shaker.

DLT of the Sweethearts - Miraculin and Brazzein (and B21)

Digestion Reactions
Miraculin Brazzein B21
DNA 6 11 7
FD Buffer (10x) 2 2 2
diH2O 10 5 9
EcoRI 1 1 1
SpeI 1 1 1

Placed in 37°C waterbath for 20 minutes.

Nanodrop Specs
Name Concentration (ng/μL)
Miraculin 9.5
Brazzein 10.2
B21 Vector (V0120) 8.4


Ligation Reactions
Miraculin Brazzein Control
Insert 3.5 0.75 0
Vector 6 6 6
Buffer (10x) 2 2 2
Ligase 1 1 1
H2O 6.5 10.25 0
  • The ligation of Miraculin was done at a 3:1 ratio (insert to vector) due to its larger size (~700 bp)
  • The ligation of Brazzein was done at a 2:1 ratio due to its smaller size (~200 bp)



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