Difference between revisions of "Team:Paris Saclay/Notebook/October/3"

(→‎Monday 3rd October)
(→‎Lab work)
 
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= Monday 3<sup>rd</sup> October=
 
= Monday 3<sup>rd</sup> October=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
−
====Colony PCR of X clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)====
+
====Colony PCR of 32 clones containing GFP 1.9 in pSB1C3 (pPS16_020)====
−
''By Maxence''
+
''By Maxence & Caroline''
  
−
For that purpose, X clones were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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 that purpose, 32 clones were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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 :
 
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
Line 35: Line 35:
 
|-
 
|-
 
|72°C
 
|72°C
−
|1 min
+
|30 sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
Line 51: Line 51:
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!Clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)
+
!Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
 
|-
 
|-
 
|Primers
 
|Primers
Line 67: Line 67:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|FRB - GFP 11 - FKBP - GFP 10
+
|GFP 1.9
−
|1714
+
|1135
 
|}
 
|}
  
−
GEL
+
[[File:T--Paris Saclay--Gel11111116.png|400px|thumb|center|Result of the migration]]
  
−
====Colony PCR of X clones containing GFP 1.9 in pSB1C3 (pPS16_020)====
+
[[File:T--Paris Saclay--Gel11111117.png|400px|thumb|center|Result of the migration]]
−
''By Maxence''
+
  
−
For that purpose, X clones were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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.
+
====Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1====
 +
''By Maxence & Caroline''
  
−
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
+
Extracted pPS16_009 from the 2nd October was digested by several restriction enzymes in order to verify if the template we used was the good one.
−
* 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:  
+
For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following:
−
{| class="wikitable"
+
−
|-
+
−
!Step
+
−
!Temperature
+
−
!Time
+
−
|-
+
−
|Initial denaturation
+
−
|95°C
+
−
|3 min
+
−
|-
+
−
|rowspan="3"|30 cycles
+
−
|95°C
+
−
|30 sec
+
−
|-
+
−
|48.4°C
+
−
|30 sec
+
−
|-
+
−
|72°C
+
−
|30 sec
+
−
|-
+
−
|Final Extension
+
−
|72°C
+
−
|7 min
+
−
|-
+
−
|Hold
+
−
|4°C
+
−
|$\infty$
+
−
|}
+
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
* 5 µL of GFP 1.9 (pPS16_009) clone 1
 +
* 2 µL of buffer orange
 +
* 2 µL of restriction enzyme NdeI
 +
* 11 µL of water
  
−
{| class="wikitable"
+
Furthermore, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes BsaBI as following:
−
|-
+
−
!Matrix
+
−
!Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
+
−
|-
+
−
|Primers
+
−
|iPS168 and iPS169
+
−
|-
+
−
|}
+
  
−
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.
+
* 5 µL of GFP 1.9 (pPS16_009) clone 1
 +
* 2 µL of buffer orange
 +
* 2 µL of restriction enzyme BsaBI
 +
* 11 µL of water
  
−
PCR products expected were :
+
The mix were incubated for 30 minutes at 37°C.
  
−
{| class="wikitable"
+
====Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6====
−
|-
+
''By Maxence & Caroline''
−
!PCR products
+
 
−
!Expected band size (bp)
+
Extracted pPS16_019 and pPS16_018 from the 2nd October were digested by restriction enzymes EcoRI in order to verify the concentration as following:
−
|-
+
 
−
|GFP 1.9
+
* 5 µL of plasmid
−
|1135
+
* 2 µL of buffer FD
−
|}
+
* 2 µL of restriction enzyme EcoRI
 +
* 11 µL of water
 +
 
 +
The mix were incubated for 15 minutes at 37°C.
 +
 
 +
====Gel of digested products====
 +
''By Maxence & Caroline''
 +
 
 +
After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
  
−
GEL
+
[[File:T--Paris Saclay--Gel11111118.png|400px|thumb|center|Result of the migration]]
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:53, 14 October 2016

Monday 3rd October

Visualization

Colony PCR of 32 clones containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence & Caroline

For that purpose, 32 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
48.4°C 30 sec
72°C 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
Primers iPS168 and iPS169

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 1135
Result of the migration
Result of the migration

Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1

By Maxence & Caroline

Extracted pPS16_009 from the 2nd October was digested by several restriction enzymes in order to verify if the template we used was the good one.

For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following:

  • 5 µL of GFP 1.9 (pPS16_009) clone 1
  • 2 µL of buffer orange
  • 2 µL of restriction enzyme NdeI
  • 11 µL of water

Furthermore, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes BsaBI as following:

  • 5 µL of GFP 1.9 (pPS16_009) clone 1
  • 2 µL of buffer orange
  • 2 µL of restriction enzyme BsaBI
  • 11 µL of water

The mix were incubated for 30 minutes at 37°C.

Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6

By Maxence & Caroline

Extracted pPS16_019 and pPS16_018 from the 2nd October were digested by restriction enzymes EcoRI in order to verify the concentration as following:

  • 5 µL of plasmid
  • 2 µL of buffer FD
  • 2 µL of restriction enzyme EcoRI
  • 11 µL of water

The mix were incubated for 15 minutes at 37°C.

Gel of digested products

By Maxence & Caroline

After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Result of the migration





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