IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/29

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Team Allergy
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

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

Transformation ( see transformation protocol)

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

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)

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 for 8 hours. 2 mL of LB+AMP was added to the cultures, bring the total volume to 5 mL of LB+AMP. Cultures were left to shake overnight.

DLT of the Sweethearts - Miraculin and Brazzein (and B21)
Placed in 37°C waterbath for 20 minutes.

Digestion Gel



Gel Lanes: 1. 1 kb plus ladder 2. Miraculin digested with EcoRI/SpeI 3. Brazzein digested with EcoRI/SpeI 4. B21 digested with EcoRI/SpeI 5. 1 kb plus ladder


 * 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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