Team:Freiburg/Labjournals/Cloning/September
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)
- Qiagen kit
- eluted in 20µl dH2O
- concentration: 6.4ng/µl
Ligation of pOP+BBa_I13504 (JN)
- Backbone: 3.4 µl
- BBa_I13504: 10.5 µl
- buffer: 2 µl
- T4 ligase: 1 µl
- dH2O: 3.1 µl
Trafo: Ligation of pOP BBA_I13504 in E.coli T10 (LS)
- 7 µl 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
PCR clean-up (JN)
- Qiagen kit
- eluted in 20 µl dH2O
1 | 7.6 ng/µl |
2 | 9.2 ng/µl |
3 | 7.2 ng/µl |
Digest: K592009 with PstI (JN)
amount | ingredient |
---|---|
25µl | DNA |
1µl | PstI |
5µl | buffer 3.1 |
19µl | dH20 |
- incubation at 37°C for 1h
- analysis on 1% agarose gel
Gel-ex: Digest of K592009 and pOP (LS)
- qiagen kit
- eluted in 20µl
- pOP: 3,9 ng/µl
- K59209: 9,0 ng/µl
Ligation: K592009 into pOP (LS)
ingredient | amount [µl] |
---|---|
pOP | 12,83 |
K592009 | 2,33 |
T4 ligase buffer | 2 |
T4 ligase | 1 |
dH2o | 2 |
- incubation at Rt for 1h
Transformation of ligation into E.coli T10 (LS)
- 7µl of ligation mix
- transformation according to protocol
- plated on LB-Amp
- incubation at 37°C o/n
2015/09/10
Clean-up of pOP EcoRI digest (JN)
- Qiagen kit
- eluted in 20µl dH2O
- concentration: 1.8ng/µl
Subsequent digest with PstI (JN)
ingredient | amount [µl] |
---|---|
DNA | 16 |
PstI | 1 |
buffer 3.1 | 5 |
dH2O | 28 |
- 37°C for 1h
- analysis on 1% agarose gel, see below
Digest of pOP_Anderson_GFP with EcoRI (JN)
ingredient | amount [µl] |
---|---|
DNA | 5 |
EcoRI | 1 |
CutSmart | 2 |
dH2O | 12 |
- 37°C for 1h
Clean-up of pOP_A_GFP EcoRI digest (JN)
- Qiagen kit
- eluted in 20µl dH2O
- concentration: 28.0 ng/µl
Subsequent digest with PstI (JN)
ingredient | amount [µl] |
---|---|
DNA | 16 |
PstI | 1 |
buffer 3.1 | 5 |
dH2O | 28 |
- 37°C for 1h
- analysis on 1% agarose gel
Gel-ex of pOP digested with EcoRI and PstI (JN)
- Qiagen kit
- eluted in 20µl dH2O
- concentration: 9.6 ng/µl
2015/09/11
Liquid cultures of Ligation pOP + BBa_K592009 (JN)
- 10 colonies were picked
- inoculated in 5ml LB-Amp each
- 37°C, shaking, until evening
OD of liquid cultures (JN)
- OD of the liquid cultures from the morning was measured
- cultures were diluted to a OD of 0.4 in a volume of 5ml and grown again for half an hour
- induction with 0.5µl of IPTG
- incubated o/n shaking at 37°C
2015/09/12
Mini-Prep of 3 induced cultures of pOP_K592009 (JN)
- unfortunately, no liquid were blue as they should be if expressing the protein
- Zymo Research kit
- eluted in 30µl dH2O
concentration [ng/µl] | |
---|---|
pOP_K592009 1 | 48.7 |
pOP_K592009 4 | 46.5 |
pOP_K592009 7 | 52.7 |
Test-digest of pOP_K592009 with EcoRI (JN)
ingredient | amount [µl] |
---|---|
DNA | 15 |
EcoRI | 1 |
CutSmart | 2 |
dH2O | 2 |
- 37°C for 1h
Clean-up of pOP_K592009 EcoRI digest (JN)
- Qiagen kit
- eluted in 20µl dH2O
concentration [ng/µl] | |
---|---|
pOP_K592009 1 | 20.0 |
pOP_K592009 4 | 14.0 |
pOP_K592009 7 | 13.7 |
Subsequent digest with PstI (JN)
ingredient | amount [µl] |
---|---|
DNA | 16 |
PstI | 1 |
buffer 3.1 | 2 |
dH2O | 1 |
- 37°C for 1h
- analysis on 1% agarose gel, band fot the backbone was clearly visible at the right height but no band for the insert was visible
Digest: BBa_I13504 with EcoRI (LS)
ingredients | volume |
---|---|
BBa_I13504 | 10µl |
CutSmart | 4µl |
EcoRI | 1µl |
dH2O | 25µl |
- incubation at 37°C for 1h
- clean-up with PCR-Clean-up kit (Roche)
- eluted in 20µl: 34,5ng/µl
Digest: Clean-up (BBa_I13504) with PstI (LS)
ingredients | volumes |
---|---|
BBa_I13504 (EcoRI digested) | |
buffer 3.1 | 4µl |
PstI | 1µl |
dH20 | 17µl |
- incubation at 37°C for 1h
- analysis on 1% agarose gel, correct band of the BioBrick was cut out of the gel
Gel-ex of BBa_I13504 (JN)
- Qiagen kit
- eluted in 20µl dH2O
- concentration: 9.1ng/µl
Ligation of pOP + BBa_I13504 (JN)
ingredients | volumes |
---|---|
pOP | 7µl |
BBa_I13504 | 2.9µl |
buffer | 2µl |
T4ligase | 1µl |
dH20 | 7.1µl |
- 1h at RT
Trafo of pOP_I13504 into BL21 (LS)
- 7 µl of ligation mix
- transformation according to protocol
- plated on LB-Amp
2015/09/13
Inoculation of pOP_I13504 (LS)
- inoculation of 10 x 5 ml LB-Amp with ligation clones
- incubation at 37°C
OD Measurement of liquid cultures (JN)
- OD for each liquid culture was measured
- dilution for each culture to a final OD of 0.4
- incubation at 37°C for 30min, shaking
- OD was measured again –> for all cultures about 0.5
- induction with 5µl IPTG for a final concentration of 1mM
- incubation at 37°C, shaking
Checked for fluorescence (JN/LS)
- measuring of fluorescence in all 10 cultures with UV light
- all cultures showed fluorescence
Restreaked 3 cultures of pOP_I13504 (LS)
- incubation at 37°C o/n
2015/09/14
Inoculation of pOP_I13504 (LS)
- 6 x 5ml LB-Amp, two cultures of each colony
- incubation at 37°C, shaking
OD measurement + induction with IPTG o/n (JN)
- cultures were diluted to obtain an OD of 0.3
- incubation for ca. half an hour shaking at 37°C
- OD measurement until OD of approximately 0.5 was reached
- one culture of each colony was induced with IPTG in a final concentration of 1 mM, the other was left uninduced
- incubation o/n at 30°C, shaking
2015/09/15
GFP measurement with UV light (JN)
- expressed GFP was visible with the bare eye as well as under UV light
- one liquid culture was pelleted, 1 ml for following SDS-PAGE, the rest to be prepped
SDS-PAGE (Protein Purification Labjournal JD)
- 1 ml of liquid culture was pelleted and the supernatant discarded
- 300 µl of 2.5x SDS Loading-dye was added
- boiling at 95°C for 10min, cooldown
- 20 µl were loaded on a 12.5% SDS-PAGE gel
- analysis via coomassie staining