Revision as of 09:46, 9 September 2015

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Cloning

2015/09/01

2015/09/02

Digest: pOP, pILS3 and K592009 with EcoRI (LS)

amount	ingredient
~5µl	plasmid
5µl	Cut Smart
1µl	EcoRI
up to 50µl	dH2O

incubation at 37°C, ~1h

PCR Purification of EcoRI digest (JN)

Qiagen kit
eluted in 20µl dH2O

	concentration [ng/µl]
pOP	3.3
pILS3	23.
BBa_K592009	2.3

Digest of pOP, pILS3 and BBa_K592009 with PstI (JN)

ingredient	amount [µl]
DNA	16/17/18
PstI	1
buffer 3.1	5
dH2O	28/27/26

37°C for 1h
analysis on 1% agarose gel

Gel-ex of BBa_I13504 (JN)

Qiagen kit
eluted in 20µl dH2O
concentration: 10.2ng/µl

Repetition of pOP Digest with EcoRI (JN)

ingredient	amount [µl]
DNA	5
EcoRI	1
CutSmart	5
dH2O	39

37°C for 1h

PCR Purification of pOP Digest

Qiagen kit
eluted in 20µl dH20
concentration: 3.7ng/µl

pOP digest with PstI

ingredient	amount [µl]
DNA	17
PstI	1
buffer 3.1	5
dH2O	27

37°C for 1h
analysis on 1% agarose gel

Gel-ex of pOP (JN)

Ligation of pOP+BBa_I13504 (JN)

Trafo: Ligation of pOP BBA_I13504 in E.coli T10 (LS)

10µl of ligation
transformation according to protocol

2015/09/03

Inoculation of pOP_BBa_I13504 (LS)

picked 3 clones
inoculation in LB-Amp (5ml)
incubation at 37°C

Inoculation of pOP_BBa_I13504 (JN)

10 more clones were picked and inoculated o/n
each clone in 5ml of LB-Amp

2015/09/04

Mini-Prep of pOP_Anderson_GFP (JN)

all liquid cultures were fluorescent, only three were prepped, four more were pelleted in stored in the freezer as backup
Qiagen kit
eluted in 30µl dH2O

	concentration [ng/µl]
pOP_Anderson_GFP 4	21.1
pOP_Anderson_GFP 9	42.9
pOP_Anderson_GFP 11	37.2

sample 9 and 11 were sent in for sequencing

Inoculation of pOP (JN)

inoculation of 3 colonies from already performed re-trafo (LS)
3 liquid cultures with 5ml LB-Amp medium each
inoculated o/n

2015/09/05

Miniprep: pOP (LS)

15µl of pOP
Zymo kit
elution in 65µl: 81,0 ng/µl

Digest: pOP and K592009 with EcoRI (LS)

amount	ingredient
10µl/~4µl	pOP/K592009(all that was left)
1µl	EcoRI
5µl	Cut Smart
up to 50µl	dH20

incubation at 37°C for 1h

Clean-up of Digest (LS)

Zymo PCR clean-up kit
elution in 20µl:
pOP:21,3ng/µl
K592009:2,7ng/µl

Digest: pOP and K592009 from clean-up with PstI (LS)

amount	ingredient
18µl	pOP/K592009
5µl	buffer 3.1
1µl	PstI
36µl dH2O

incubation at 37°C, 1h
analysis on 1% agarose gel:

-small band visible for pOP -nothing for K592009

Inoculation: 3x 5ml LB-Cml wit K592009 (LS)

incubation at 37°C, o/n

2015/09/06

Mini-Prep of K592009 (JN)

Qiagen kit
eluted in 20µl dH2O

1	77.0 ng/µl
2	94.4 ng/µl
3	64.6 ng/µl

Digest of all three preps with EcoRI (JN)

amount	ingredient
5µl	K592009
1µl	EcoRI
5µl	Cut Smart
39µl	dH20

incubation at 37°C for 1h

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