Team:Paris Saclay/Notebook/September/14

Wednesday 14th September

Lab work

Visualization

Glycerol stocks of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3

"By Maxence"

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

  • pPS16_009 (GFP 1.9) clone 2
  • pPS16_009 (GFP 1.9) clone 7
  • pPS16_014 (GFP 1.9) clone 8
  • pPS16_014 (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

"By Maxence"

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

  • pPS16_009 (GFP 1.9) clone 2
  • pPS16_009 (GFP 1.9) clone 7
  • pPS16_014 (GFP 1.9) clone 8
  • pPS16_014 (GFP 1.9) clone 12

Samples preparation for sequencing

By Maxence

20 µL of plasmids pSB1C3 GFP 1.9 (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.

PCR of cleaned up Ligation products (fragment 1 and fragment 2)

By Maxence

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

PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11

By Maxence

In order to obtain pZA11 containing the desired sequences, 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 gblock spacer in X gblock ST sgRNA in pUC19 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