Monday 26^th September

Visualization

Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

By Maxence, Mahnaz & Coline

A colony PCR was done for 16 clones (8 clones obtained by Gibson with 2 fragments and 8 clones obtained by Gibson with 3 fragments) from the 23rd 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:

Primers used were:

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 :

Result of the migration

Two PCR products were at the good size: clones 3 & 11 were selected for sequencing.

NanoDrop Measurements

By Maxence, Manhaz & Caroline

Samples preparation for sequencing

'By Maxence, Mahnaz & Caroline'

20 µL of plasmids dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) (clones 2, 7 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM) and iPS169 (5µM) were sent for sequencing.

Gel of extracted GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence, Manhaz & Caroline

4 µL of extracted plasmids and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

Result of the migration