Team:Heidelberg/Templates/DelH week26

From 2013.igem.org

(Difference between revisions)
(→Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest)
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5 µl Dream-Taq <br/>
5 µl Dream-Taq <br/>
4 µl ddH<sub>2</sub>O
4 µl ddH<sub>2</sub>O
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'''Results:''' <br/>
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* Colonies B4, B9, B10 and C4 were positive <br/>
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* 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/>
Line 21: Line 24:
'''Results:''' <br/>
'''Results:''' <br/>
* All three screenings were positive. Consequently our electrocompetent BL21-DE3 with DelRest and pIK8.6 really contain DelRest.
* All three screenings were positive. Consequently our electrocompetent BL21-DE3 with DelRest and pIK8.6 really contain DelRest.
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==Screening ccdB-construct==
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14 colonies were picked, we had a negative and a positive (pRB14) was used
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{| class="wikitable"
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|-
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! Reagent !! Volume
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|-
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| KH10 || 0.5 µl
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|-
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| VFII || 0.5 µ
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|-
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| ddH<sub>2</sub>O || 4 µl
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|-
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| Dream Taq || 5 µl
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|}
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Annealing temp.: 56°C, 35 cycles, elongation time= 50 s
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|}

Revision as of 23:08, 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 |}

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