Alex Leffell

Performed two LR reactions for practice

Aliquot 1:

1 uL of eYFP @ 5 fm/uL

1 uL of TRE-t @ 5fm/uL

1 uL of DEST @ 10 fm/uL

1 uL of clonase

total: 4 uL

Aliquot 2:

1 uL of eYFP @ 5 fm/uL

1 uL of HEF-1a @ 5fm/uL

1 uL of DEST @ 10 fm/uL

1 uL of clonase

let sit at room temperature overnight

-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)

performed nandrop on minipreped plasmids in order to determine their concentration
for each, calculated necessary volume to get  1ug of plasmid for a gel
created a gel