Tuesday 13th September
Lab work
Visualization
Colony PCR of 16 clones containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence & Mahnaz
A colony PCR was done for 16 clones from the 12th September. For that purpose, 16 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
- 2.5 µL DreamTaq Buffer
- 0.5 µL of dNTPs (10mM)
- 1 µL of each primer mix (10µM)
- 0.13 μl of DreamTaq Pol
PCR was performed as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
3 min
|
| 30 cycles
|
95°C
|
30 sec
|
| 72°C
|
30 sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
|
| Primers
|
iPS84 and iPS140
|
After amplification, 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)
|
| GFP 1.9 in pSB1C3
|
862
|
All PCR products were at the good size. Clones 2, 7, 8 and 12 were selected and were grown at 37°C overnight.
Gel of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) cleaned-up Ligation products
By Maxence & Mahnaz
As strange results were obtained the 12th September, gel of cleaned-up Ligation products was run with less DNA: 2 µL of each cleaned up Ligation 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
|
GEL bizare
Small bands was obtained and appeared to be at the good size, even if the results were worst for fragment 2. Ligation step appeared to work, so a new approach should be used for the amplification step.
gblock 1.1 - 1.2 and gblock 2.1 - 2.2 Ligation
By Maxence & Mahnaz
A new ligation was run due to strange results obtained the 12th September.
gBlocks pPS16_001 and pPS16_002 were ligated together as following :
- 1 µL of pPS16_001 PCR product from 9th September
- 1 µL of pPS16_002 PCR product from 9th September
- 2 µL of Buffer T4 10X
- 2 µL of ligase T4 enzyme
This strategy aims to obtain fragment 1.
gBlocks pPS16_003 and pPS16_004 were ligated together as following :
- 1 µL of pPS16_003 PCR product from 9th September
- 1 µL of pPS16_004 PCR product from 9th September
- 2 µL of Buffer T4 10X
- 2 µL of ligase T4 enzyme
This strategy aims to obtain fragment 2.
The ligation product was put at rooming temperature for 4 hours.
