Difference between revisions of "Team:Paris Saclay/Notebook/September/14"

(Created page with "= Wednesday 14<sup>th</sup> September= ==Lab work== ===Visualization=== ==== Glycerol stocks of clones 7, 9, 15 and 16 containing GFP 1.9 in pSB1C3 ==== "By Maxence" The gly...")
 
(→‎PCR of cleaned up Ligation products (fragment 1 and fragment 2))
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''By Maxence''
 
''By Maxence''
  
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With the results obtained the 12th and 13th september, a new amplification approach was tested: the Ligation products were diluated at 1/10 and two buffer (HF anf GC) were tested with others different PCR conditions.
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With the results obtained the 12th and 13th september, a new amplification approach was tested: the Ligation products were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions.
  
 
For each 50μl of reaction, mix the following reagents :
 
For each 50μl of reaction, mix the following reagents :

Revision as of 20:26, 13 September 2016

Wednesday 14th September

Lab work

Visualization

Glycerol stocks of clones 7, 9, 15 and 16 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 7
  • pPS16_009 (GFP 1.9) clone 9
  • pPS16_014 (GFP 1.9) clone 15
  • pPS16_014 (GFP 1.9) clone 16

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

A MODIFIER

Plasmids extraction of clones 7, 9, 15 and 16 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 7
  • pPS16_009 (GFP 1.9) clone 9
  • pPS16_014 (GFP 1.9) clone 15
  • pPS16_014 (GFP 1.9) clone 16

Samples preparation for sequencing

By Maxence

20 µL of plasmids pSB1C3 GFP 1.9 (clones 7, 9, 15 and 16) were sent to be sequenced. 20 µL of the primers iPS83 (5µM) and iPS84 (5µM) were sent for sequencing.

A VERIFIER

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 were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions.

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