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Team:MIT bconst
From 2010.igem.org
iGEM 2010
bacterial biobrick construction |
The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes. |
General Biobrick Construction Protocol Miniprep. To extract DNA from cells, we used the QIAprep Spin Miniprep Kit according to protocol. Virtual Restriction Mapping. The sequences of all our individual parts were entered into a shared Geneious library, and all of our Biobrick parts were created virtually parallel to actual construction. We used Geneious to find enzymes for restriction mapping and sequencing validation of parts. DNA Digestion. For both restriction mapping and for digesting a plasmid to insert or extract a biobrick, the appropriate buffer was added 1:10 to a DNA sample and 10 units of the appropriate enzyme(s) were used per 1ug of DNA. The reactions were allowed to run at the temperature optimal for the enzyme for 1 hour, the enzymes were denatured at 65°C for 20 minutes, and then the reactions were held at 16°C. Antarctic Phosphatase. To prevent recircularization of digested plasmids, we used NEB's Antarctic Phosphatase according to protocol. Gel Electrophoresis. To view lengths of DNA, Orange G 6x from NEB was added 1:3 to 300ng miniprepped (and sometimes digested) DNA and run against 5uL of Hyperladder I from NEB in the lanes of 1% UltraPure agarose (Invitrogen) gels in TAE buffer at 120V for 45 minutes. DNA Gel Extraction. To extract the DNA from the agarose gel, the QIAquick Gel Extraction kit was used according to protocol. Transformation. Aliquots of competent JM2.300 E.coli cells were thawed on ice for 8 minutes. Approximately 10ng of each plasmid were added to the competent cells and the tube was tapped to mix. The cells incubated on ice for 30 minutes after which they were heat shocked at 42°C for exactly 30 seconds and then incubated on ice for 2 minutes. Each transformation received 0.9 mL of room temperature Super Optimal broth with Catabolite repression, and were shaken at 37°C at 280 rpm for 60 minutes. On a LB plate with antibiotics that select for cells transformed with our DNA, 20 uL of the mixture was plated on one half, the rest of the cells were pelleted and plated on the other half. Plates incubated upright for 20 minutes then agar up for 12-16 hours at 37°C. |
