3/7/2014
3/14/14
-In order the practice GIbson Assemblies, in a tube I put:
4.2 uL of o55 (1000ng/ 237 ng/L = 4.2 uL)
12.8 uL of H2O (17-4.2)
2uL of buffer
1 uL of PAC-1 restriction enzyme
-In order to prepare a gel in order to test the assemblies, I combined:
1g agarose
100 mL H20
microwaved until boiling, then heated intermittently and briefly until fully dissolved. Solution was then allowed to cool.
After cool, I added 10 uL of sybrsafe dye to agarose solution
poured gel into cast, up to just under height of comb. ( pour extra cast because UV light for imaging damages DNA)
-In order the extract the plasmids from our transformed bacteria, I performed a miniprep.
2ml of luria broth/ bacteria suspension was centrifuged for 3 min @ 6000g. Then I poured out the broth.
I added 250 uL of P1 buffer to each culture pellet and mixed by repipetting. This resuspended the cells
I added 250 uL of P2 buffer to lyse the cells. Very gently invert.
I added 350 uL of N3 buffer to halt lysation. Invert gently until blue color disappears.
Centrifuge for 10 min @ max rpm
( I left at this point, but protocol from miniprep kit was followed)