From 2008.igem.org
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Revision as of 18:24, 16 September 2008
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Construction of rbs-TetR-mRFP-LVA-tripart (L173)
   
PCR Screening of L173 transformants
Transformation results (rbs-TetR in mRFP-LVA-tripart-pSB1A3)
Ligation L173
- insert: D112 (ES) rbs-TetR
- vector: D187 (EX) mRFP-LVA-tripart-pSB1A3
| Sample
| positive control
pUC19
| negative control
no DNA
| ligation control
(without insert)
| L173 (3:1 ratio)
| L173 (4:1 ratio)
|
| Number of colonies
| many
| 1
| 0
| 2
| 0
|
PCR Screening
PCR screening programm
- elongation tim: 2 min
- primers used: O18 & O19
- positive PCR control: S158 (pSB3K3)
- negative PCR control: no template
| Well n°
| 1
| 2
| 3
| 4
| 5
| 6
| 7
|
| Sample
| 1 kb
DNA ladder
| positive PCR
control
| negative PCR
control
| negative transformation
control clone
| L173.1
| L173.2
| 100 bp
DNA ladder
|
| Expected size
| 1894 bp
|
| Measured size
| 1,2 kb
| 1,2 kb
|
Results: the clones are not correct.
Digestion
| Digestion n°
| DNA substrat
| Digestion by
|
| D112
| MP106: S03879 (rbs-TetR) in pSB1A2
| EcoRI & SpeI
|
- 5 µL of DNA
- 3 µL of 10X buffer n°2
- 0,3 µL 100X BSA
- 1 µL of EcoRI
- 1 µL of SpeI
- 19,7 µL of water
Incubation 2h30 at 37°C and then 20 min at 65°C.
Not purified yet. Put at -20°C.
Construction of pFlhB-mRFP-LVA-tripart & pFliL-ECFP-LVA-tripart
- L183 = pFlhB - mRFP LVA+
- L184 = pFliL - ECFP LVA+
- L185 = pFliL - ECFP LVA-
Ligation
Protocol
| Ligation Name
| Vector Name
| Volume Vector (µL)
| Insert
| Volume Insert (µL)
|
| L183
| D187
| 2,5
| D134
|
|
| L184
| D198
| 1,11
| D149
|
|
| L185
| D199
| 0,65
| D149
|
|
|
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