Monday 12^th September

Visualization

Glycerol stocks of clones 4, 9, 13 and 16 containing FRB - GFP11 in pSB1C3 (pPS16_019)

"By Maxence & Mahnaz"

The glycerol stock of the bacteria with the following plasmids were made.

• pPS16_019 (FRB - GFP11) clone 4
• pPS16_019 (FRB - GFP11) clone 9
• pPS16_019 (FRB - GFP11) clone 13
• pPS16_019 (FRB - GFP11) clone 16

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 4, 9, 13 and 16 containing FRB - GFP11 in pSB1C3 (pPS16_019)

"By Maxence & Mahnaz"

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

• pPS16_019 (FRB - GFP11) clone 4
• pPS16_019 (FRB - GFP11) clone 9
• pPS16_019 (FRB - GFP11) clone 13
• pPS16_019 (FRB - GFP11) clone 16

Samples preparation for sequencing

By Maxence & Mahnaz

20 µL of plasmids pSB1C3 FRB - GFP 11 (pPS16_019) (clones 4, 9, 13 and 16) were sent to be sequenced. 20 µL of the primers iPS83 (5µM) and iPS84 (5µM) were sent for sequencing.

NanoDrop Measurements

By Maxence & Mahnaz

Sample	Concentration (ng/µL)
FRB - GFP 11 clone 4	96.29
FRB - GFP 11 clone 9	35.51
FRB - GFP 11 clone 13	79.91
FRB - GFP 11 clone 16	50.58
PCR product GFP 1.9 from 9th September	438.11
pSB1C3 from 22nd August	31.61
PCR fragment gblock 1.1 from 9th September	239.41
PCR fragment gblock 1.2 from 9th September	195.92
PCR fragment gblock 2.1 from 9th September	165.99
PCR fragment gblock 2.2 from 9th September	143.45

Gibson of cleaned up PCR products GFP 1.9 from 9th September x pSB1C3 treated by DpnI from 22nd August

By Maxence & Mahnaz

Gibson was performed with cleaned up PCR products GFP 1.9 (insert) (obtained the 9th September) x pSB1C3 treated by DpnI (plasmid) (obtained the 22nd August) with the following protocol:

For each 20μl of reaction, mix the following reagents :

• 0.2 µL of insert
• 3.16 µL of plasmid
• 6.64 µL of water
• 10 µL of buffer mix

Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.

Transformation of DH5a cells with GFP 1.9 in pSB1C3 (pPS16_020) obtained by Gibson

By Maxence & Mahnaz

Dh5a cells were transformed with pSB1C3 containing GFP 1.9, or controls (no buffer mix and plasmid alone) using the usual protocol.

gblock 1.1 - 1.2 and gblock 2.1 - 2.2 Ligation

By Maxence & Mahnaz

gBlocks pPS16_001 and pPS16_002 were ligated together as following :

• 8 µL of pPS16_001 PCR product from 9th September
• 8 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 :

• 8 µL of pPS16_003 PCR product from 9th September
• 8 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 2 hours.

Clean-up of Ligation products

By Maxence & Mahnaz

Ligation products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

NanoDrop Measurements

By Maxence & Mahnaz

Sample	Concentration (ng/µL)
gblock 1.1 and gblock 1.2 cleaned-up Ligation product	194
gblock 2.1 and gblock 2.2 cleaned-up Ligation product	157

PCR of cleaned up Ligation products (fragment 1 and fragment 2) with 3% DMSO

By Maxence & Mahnaz

As the annealing temperature (Tm) seems too high to obtain good results for fragments 1 and 2 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. 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 (5X)
• 0,5 µL of Phusion polymerase
• 31 µL of nuclease free water
• 1.5 µL of DMSO

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
	60°C	30sec
	72°C	1 min
Final Extension	72°C	2min
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 PCR products

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)
Fragment 1	1920
Fragment 2	1831

A large smearing in agarose gel of PCR products was obtained. As this result was not usable, a new PCR was run by diluting the Ligations products : 1/100, 1/10,000 and 1/1,000,000.

A large smearing in agarose gel of PCR products was also obtained, but the smears were seemed to decrease with the dilution rate.