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Revision as of 07:59, 10 September 2015
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
