Wednesday 24th August
Lab work
Visualization
Gel Electrophoresis of Gibson Assembly's products(fragment 3-4) and GFP 1-9 cloned in PSB1C3
By Terrence
Colony PCR of 12 colonies transformed with Gibson Assembly products (fragment 3-4).
Colony PCR of 12 colonies containing the GFP 1-9.
- Clones 2, 4, 7 and 11 are as expected.
pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction
By Terrence
The extraction was carried out following the usual protocol.
Nano drop :
|
|
concentration (ng/µL)
|
260/230
|
260/280
|
| 1.2.14
|
47.81
|
1.64
|
1.84
|
| 1.2.8
|
834.5
|
2.40
|
1.99
|
| SgRna Nm 12
|
124.21
|
2.00
|
1.89
|
Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET
By Terrence
First we made a ligation with
| Component
|
Volume (µL)
|
| Reaction Buffer
|
10
|
| PCR Product (FRB or Nm)
|
1
|
| pJET1.2/blunt
|
1
|
| water, nuclease free
|
7
|
| T4 DNA Ligase
|
1
|
Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the usual protocol.
Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control.
By Léa & Manhaz
We have decided to use the plasmid coding dCas9 Nm to amplify the fragement corresponding g-block 1.2.
200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water.
The plasmid then reserved in -20.
PCR Q5 on plasmid
By Léa & Manhaz
Plasmid pJet cotnains gblock 1.2 (confirmed by sequencing) was used as template to amplify the fragement 1.2.
also we amplify the plasmids extracted from 2 colonies contrain the plasmid coding dCas Nm ordered from adgene.
Q5 PCR was performed on plasmids with the following protocol:
For each 50μl of reaction, mix the following reagents :
- 1 µL of plasmid
- 1 µL of dNTPs (10mM)
- 1 µL of each primer mix (10µM)
- 10 µL of Q5 buffer (5X)
- 0,5 µL of Q5 high fidelity polymerase
- 35,5 µL of nuclease free water
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
Perform PCR as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
98°C
|
30sec
|
| 30 cycles
|
98°C
|
10sec
|
| Tm
|
20sec
|
| 72°C
|
t
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infinity\$
|
Primers used were:
| Matrix
|
gblock1.2 in pJET
|
plasmid adgene contain dCas9 Nm clones 1 and 2
|
| Primers
|
iPS121 and iPS122
|
iPS121 and iPS122
|
| Tm
|
72°C
|
72°C
|
| t
|
30 sec
|
30 sec
|
NanoDrop Measurements
By Mahnaz
| Sample
|
Concentration (ng/µL)
|
| PCR fragment 1.2
|
69.08
|
gBlock 1.1 and gblock 1.2 Ligation
By Mahnaz
gBlock 1.1 and gblock 1.2 were ligated together as following :
- 8µL of gBlock 1.1 PCR product from the 04/08/2016
- 8µM of gblock 1.2 PCR product from 24/08/2016
- 2µL of Buffer T4 10X
- 2µL of ligase T4 enzyme
The ligation product was put at 4°c overnight.
