IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/29
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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.
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
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
Transformed amiRNA into turbo cells and plated on LB amp plates--will check for colonies tommorow (Plated at ~ 4pm)
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.
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