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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

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3/14/14

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\-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)

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3/16

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}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{cloak}