From 2008.igem.org
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Revision as of 18:00, 4 September 2008
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← Yesterday ↓ Calendar ↑Tomorrow →
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← Yesterday ↓ Calendar ↑Tomorrow →
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)
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| Number of colonies
| many
| 1
| 0
| 2
| 0
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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
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| Sample
| 1 kb
DNA ladder
| positive PCR
control
| negative PCR
control
| negative transformation
control clone
| L173.1
| L173.2
| 100 bp
DNA ladder
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| Expected size
| 1894 bp
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| Measured size
| 1,2 kb
| 1,2 kb
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Results: the clones are not correct.
Digestion
| Digestion n°
| DNA substrat
| Digestion by
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| D112
| MP106: S03879 (rbs-TetR) in pSB1A2
| EcoRI & SpeI
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- 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 2 hours at 37°C
Transformation results
L177 = d134 + d187 (pFlhB-mRFP LVA+)
L175 = d149 + d° (pFliL - ECFP LVA+)
L176 = d149 + d° (pFliL - ECFP LVA-)
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