Page History

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