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Team:Heidelberg/Templates/DelH week26
From 2013.igem.org
(Difference between revisions)
(→Screening ccdB-construct) |
(→Screening colonies of pSB4K5+DelH (without promotor)) |
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0.5 Primer (FS73 and FS95) <br/> | 0.5 Primer (FS73 and FS95) <br/> | ||
5 µl Dream-Taq <br/> | 5 µl Dream-Taq <br/> | ||
| - | 4 µl ddH<sub>2</sub>O | + | 4 µl ddH<sub>2</sub>O <br/> |
'''Results:''' <br/> | '''Results:''' <br/> | ||
* Colonies B4, B9, B10 and C4 were positive <br/> | * Colonies B4, B9, B10 and C4 were positive <br/> | ||
* Those colonies were inoculated in 6 ml LB Kan | * Those colonies were inoculated in 6 ml LB Kan | ||
| + | |||
==Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest== | ==Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest== | ||
Screening of plasmid pFSN-D8w (positive control), Triple clone and electrocompetent BL21-DE3 with DelRest and pIK8.6 <br/> | Screening of plasmid pFSN-D8w (positive control), Triple clone and electrocompetent BL21-DE3 with DelRest and pIK8.6 <br/> | ||
Revision as of 23:10, 21 October 2013
Contents |
21-10-2013
Preparation of large plasmids
6 ml of clones A1, A4, A7, B4
Restriction Digest of Preps with PvuI
0.5 µl PvuI-HF, 2 µl CutSmart, 1 µl 1:10 Prep (1-2 µg)
2 hours of incubation
expected bands: 11.6 kbp, 5.8 kbp, 4.6 kbp
--> only clone A1 was positive
--> A1 was send to sequencing with primer DN07
Screening colonies of pSB4K5+DelH (without promotor)
46 colonies
0.5 Primer (FS73 and FS95)
5 µl Dream-Taq
4 µl ddH2O
Results:
- Colonies B4, B9, B10 and C4 were positive
- Those colonies were inoculated in 6 ml LB Kan
Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest
Screening of plasmid pFSN-D8w (positive control), Triple clone and electrocompetent BL21-DE3 with DelRest and pIK8.6
0.5 µl FS_14
0.5 µl VR
4 µl ddH2O
5 µl Dream Taq
Results:
- All three screenings were positive. Consequently our electrocompetent BL21-DE3 with DelRest and pIK8.6 really contain DelRest.
Screening ccdB-construct
14 colonies were picked, we had a negative and a positive (pRB14) was used
| Reagent | Volume |
|---|---|
| KH10 | 0.5 µl |
| VFII | 0.5 µ |
| ddH2O | 4 µl |
| Dream Taq | 5 µl |
Annealing temp.: 56°C, 35 cycles, elongation time= 50 s
