Wednesday 14th September
Visualization
Glycerol stocks of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 (pPS16_020)
"By Maxence & Mahnaz"
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_020 (GFP 1.9) clone 2
- pPS16_020 (GFP 1.9) clone 7
- pPS16_020 (GFP 1.9) clone 8
- pPS16_020 (GFP 1.9) clone 12
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 (pPS16_020)
"By Maxence & Mahnaz"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_020 (GFP 1.9) clone 2
- pPS16_020 (GFP 1.9) clone 7
- pPS16_020 (GFP 1.9) clone 8
- pPS16_020 (GFP 1.9) clone 12
Samples preparation for sequencing
By Maxence & Mahnaz
20 µL of plasmids pSB1C3 GFP 1.9 (pPS16_020) (clones 2, 7, 8 and 12) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.
NanoDrop Measurements
By Maxence & Mahnaz
| Sample
|
Concentration (ng/µL)
|
| GFP 1.9 clone 2
|
24.84
|
| GFP 1.9 clone 7
|
95.16
|
| GFP 1.9 clone 8
|
145.6
|
| GFP 1.9 clone 12
|
42.18
|
PCR of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products
By Maxence & Mahnaz
With the results obtained the 12th and 13th September, a new amplification approach was tested: the Ligation products from the 12th were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions. Furthermore, ligations products from the 13th September were also amplified.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer (5X)
- 0,5 µL of Phusion polymerase
- 32.5 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
|
95°C
|
30sec
|
| 25 cycles
|
95°C
|
30sec
|
| 50°C
|
30sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Products from Ligation of gblock 1.1 and gblock 1.2 (fragment 1)
|
Products from Ligation of gblock 1.1 and gblock 1.2 (fragment 2)
|
| Primers
|
iPS140 and iPS122
|
iPS 123 and iPS84
|
Gel of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products
By Maxence & Mahnaz
3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| Fragment 1
|
1920
|
| Fragment 2
|
1831
|
The results were the same as previous ones.
Another strategy was to make a PCR directly of fragments 1 and 2 obtained previously even if these fragments were not working for Gibson. Fragments 1 and 2 from the 2nd September were amplified with the same protocol as mentioned above.
PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11
By Maxence & Mahnaz
In order to obtain pZA11 containing the desired sequences (pPS16_025), gblocks were amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer HF (5X)
- 0,5 µL of Phusion polymerase
- 32.5 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
|
30sec
|
| 55°C
|
30sec
|
| 72°C
|
t
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
gblock detection in pUC19 (pPS16_016)
|
gblock spacer in pUC19 (pPS16_015)
|
gblock ST sgRNA in pUC19 (pPS16_012)
|
gblock NM sgRNA in pJET
|
extract of pzA11
|
| Primers
|
iPS153 and iPS154
|
iPS155 and iPS156
|
iPS157 and iPS158
|
iPS159 and iPS160
|
iPS161 and iPS162
|
| t
|
20sec
|
20sec
|
20sec
|
20sec
|
30sec
|
PCR Clean-up of PCR products
By Maxence & Mahnaz
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Gel of cleaned up PCR products
By Maxence
After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| gblock detection
|
1020
|
| gblock spacer
|
900
|
| gblock ST sgRNA
|
310
|
| gblock NM sgRNA
|
362
|
| pZA11
|
2125
|
All PCR products were at the good size but there were no results obtained for NM sgRNA.
Another PCR was run in order to obtain PCR products from NM sgRNA, the good TM was used (63°C).
Linearization of cleaned-up PCR product pZA11
By Maxence & Mahnaz
PCR product PZA11 has been linearized for further Gibson application by using DpnI treatment :
- 30 µL of cleaned up PCR product GFP 11 - pSB1C3
- 4 µL of fast digest buffer
- 1 µL of DpnI
- 5 µL of water
The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.
PCR of pSB1C3 from dCas9 ST - GFP 11 (pPS16_017) clone 8
By Maxence & Mahnaz
In order to obtain FKBP - GFP 10 in pSB1C3 (pPS16_018) by Gibson, pSB1C3 must be amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer HF (5X)
- 0,5 µL of Phusion polymerase
- 32.5 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
|
| 67°C
|
30sec
|
| 72°C
|
1min
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
dCas9 ST - GFP 11 (pPS16_017) clone 8
|
| Primers
|
iPS148 and iPS172
|
PCR Clean-up of PCR products pSB1C3
By Maxence & Mahnaz
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Gel of cleaned up PCR products pSB1C3
By Maxence & Mahnaz
After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| pSB1C3
|
2070
|
PCR products were at the good size.
