|
← Yesterday ↓ Calendar ↑Tomorrow →
Constructions
Results of the transformation, we did yesterday
| Ligation n°
| Insert
| Vector
| Construction
| Antibio
| Number of colonies
|
| positive control
|
|
| puc 19
| Amp
| +++
|
| L173
| D112
| D187
| rbs-tetR-mRFP-LVA+
| Amp
| 0
|
| L183
| D134
| D187
| pFlhB - mRFP LVA+
| Amp
| 0
|
| L184
| D149
| D198
| pFliL - ECFP LVA+
| Amp
| 0
|
| L185
| D149
| D199
| pFliL - ECFP LVA-
| Amp
| 0
|
=>This is a supplementary time that the experiment of ligation-transformation don't work.
It means that on of this step have a mistake.
So we need to do several test to understand where the problem is located.
We Hypothesis that the problem is due to a defectious Ligase.
Checking our ligases
Since our ligation experiments didn't work during these few days, we presume that our problem came from the ligases. That's why we decided to check the activity of the ligases in our possession.
To do so:
- we will digest a plasmid with only one restriction enzyme (EcoRI).
- then we will try to ligate it back using our ligases.
Condition tested
 Before gel excision well n°4: undigested plasmid (MP101.1) well °6, 7, 8, 9: digested plasmid (D202)
| ligase
| incubation time
| amount of enzyme
|
| L1 (ligase 1)
| 2h00
| 2 U (unit)
|
| L1
| 2h00
| 400 U
|
| L1
| O/N
| 2 U
|
| L1
| O/N
| 400 U
|
| L2 (ligase 2)
| 2h00
| 2 U
|
| L2
| 2h00
| 400 U
|
| L2
| O/N
| 2 U
|
| L2
| O/N
| 400 U
|
Digestion
Reaction mixture
(carried out four times )
- ADN (MP101.1): 3.8 µL
- H2O: 21.9 µL
- 10X Buffer: 3 µL
- BSA: 0.3 µl
- EcoRI: 1 µL
Purification
- We purifed our digestion products by gel extraction (Qiagen kit)
- After elution, we obtained [DNA] ~ 25 ng/µL (based on the intensity of the band)
Overnight ligation (16°C)
- DNA: 4 µL
- 10X Buffer: 2 µL
- Ligase: 1 µL
- H2O: 13 µL
|
"
