Difference between revisions of "Team:Paris Saclay/Notebook/October/10"

(→‎Protein extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594)
(→‎Digestion of extracted plasmids by EcoRI)
 
(17 intermediate revisions by 3 users not shown)
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= Monday 10<sup>th</sup> October=
 
= Monday 10<sup>th</sup> October=
−
==Lab work==
+
 
 +
===Visualization===
 +
 
 +
====Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and BbaB0015====
 +
''By Sylvie''
 +
 
 +
The OD were measured for each overnight cultures:
 +
 
 +
{| class="wikitable center"
 +
|-
 +
!Sample
 +
!OD (1 ml)
 +
|-
 +
|FRB - GFP 11 in pSB1C3 (pPS16_019)
 +
|3
 +
|-
 +
|FKBP - GFP 10 in pSB1C3 (pPS16_018)
 +
|3.9
 +
|-
 +
|GFP 1.9 in pUC19 (pPS16_009)
 +
|4.2
 +
|-
 +
|BbaB0015
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|5
 +
|-
 +
|}
 +
 
 +
 
 +
Then the plasmids were extracted using a [[Team:Paris_Saclay/Experiments#Plasmid_DNA_extraction|"standard Plasmid Miniprep"]].
 +
 
 +
====Digestion of extracted plasmids by EcoRI====
 +
''By Maxence & Caroline''
 +
 
 +
Extracted pPS16_019, pPS16_018, pPS16_009 and BbaB0015 from the 10th October were digested by restriction enzymes EcoRI in order to verify the concentration as following:
 +
 
 +
* 2 µL of plasmid
 +
* 2 µL of buffer FD
 +
* 2 µL of restriction enzyme EcoRI
 +
* 14 µL of water
 +
 
 +
The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 
 +
[[File:T--Paris Saclay--Gelsylvie1.png|400px|thumb|center|Result of the migration]]
 +
 
 +
The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009.
 +
 
 
===BioBrick K2039000 and K2039001 characterization===
 
===BioBrick K2039000 and K2039001 characterization===
  
Line 34: Line 79:
 
|}
 
|}
  
 +
====Protein electrophoresis of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594====
 +
''By Maxence''
  
 +
A protein electrophoresis of the extracted protein was done using the [[Team:Paris_Saclay/Experiments#Protein_electrophoresis_using_the_ThermoFisher_Mini_Gel_Tank.C2.AE_device|protein electrophoresis protocol]]. For that purpose, the gel cassette Bolt 4-12% Bis-Tris Plus 10W was used and the power was set for 1 hour at 65V.
 +
 +
====Transfert of protein extracted from FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594====
 +
''By Maxence''
  
 +
The proteins on the electrophoresis gel were transfered on blotting membrane using the [[Team:Paris_Saclay/Experiments#Protein_transfert_using_the_ThermoFisher_iBlot_2_Dry_Blotting_System.C2.AE|protein transfert protocol]].
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 14:51, 18 October 2016

Monday 10th October

Visualization

Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and BbaB0015

By Sylvie

The OD were measured for each overnight cultures:

Sample OD (1 ml)
FRB - GFP 11 in pSB1C3 (pPS16_019) 3
FKBP - GFP 10 in pSB1C3 (pPS16_018) 3.9
GFP 1.9 in pUC19 (pPS16_009) 4.2
BbaB0015 5


Then the plasmids were extracted using a "standard Plasmid Miniprep".

Digestion of extracted plasmids by EcoRI

By Maxence & Caroline

Extracted pPS16_019, pPS16_018, pPS16_009 and BbaB0015 from the 10th October were digested by restriction enzymes EcoRI in order to verify the concentration as following:

  • 2 µL of plasmid
  • 2 µL of buffer FD
  • 2 µL of restriction enzyme EcoRI
  • 14 µL of water

The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Result of the migration

The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009.

BioBrick K2039000 and K2039001 characterization

Protein extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594

By Maxence

In order to perform a Western Blot, the proteins were extracted from clones (pPS16_018 clone 6, pPS16_019 clone 4, pPS16_009 clone 1 and PhB1040 strain 594) put on liquide culture the 7th October. For that purpose, the protein extraction protocol was used:

Sample OD (1 ml) Volume of lysis buffer
FRB - GFP 11 in pSB1C3 (pPS16_019) 5.1 127.5 μl
FKBP - GFP 10 in pSB1C3 (pPS16_018) 5.2 130 μl
GFP 1.9 in pUC19 (pPS16_009) 5.3 132.5 μl
PhB1040 strain 594 2.8 70 μl

Protein electrophoresis of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594

By Maxence

A protein electrophoresis of the extracted protein was done using the protein electrophoresis protocol. For that purpose, the gel cassette Bolt 4-12% Bis-Tris Plus 10W was used and the power was set for 1 hour at 65V.

Transfert of protein extracted from FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594

By Maxence

The proteins on the electrophoresis gel were transfered on blotting membrane using the protein transfert protocol.





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