Team:Paris Saclay/Notebook/August/7

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(Extraction of the Genomic DNA)
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=Thursday 7th August=
=Thursday 7th August=
==Lab work==
==Lab work==
-
===C - Salicylate Inducible Suppressing System===
+
===Salicylate Inducible Suppressing System===
-
====DNA extraction from gel agarose====
+
====DNA purification gel agarose====
-
''In progress''
+
''by Fabio''
 +
Once the segregate process made [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/6#Segregate_Process_by_Electrophoresis yesterday] by electrophoresis, the DNA correspondent to the core of '''BBa_K1372000''' was purified from the gel agarose.
-
===D - Lemon Sent===
+
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process Segregate Process Protocol]
 +
====Ligation====
 +
''by Fabio''
-
==== PCR of pCola plasmid (Geranyl Synthase)====
+
The final step to have a BioBrick Assembly is the Ligation reaction.
-
'by Melanie'
+
 
 +
<font color="#F00">TODO: illustration of the process</font>
 +
 
 +
* BioBrick '''BBa_K1372000''' (Salicylate promoter + NahR + RNA suppressor) as '''Part A'''
 +
* BioBrick '''BBa_B0015''' (Terminator) as '''Part B'''
 +
 
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Ligation BioBrick Assembly - Ligation Protocol]
 +
 
 +
===Lemon Sent===
 +
 
 +
==== PCR of pCola plasmid (Geranyol Synthase)====
 +
''by Melanie''
we use the same protocol as yesterday but we have done 8 tubes to apply a gradient in the thermocycleur during the third step:
we use the same protocol as yesterday but we have done 8 tubes to apply a gradient in the thermocycleur during the third step:
-
#57°
+
*57°
-
#56.3°
+
*56.3°
-
#55.1°
+
*55.1°
-
#53.3°
+
*53.3°
-
#51°
+
*51°
-
#49.3°
+
*49.3°
-
#47.9°
+
*47.9°
-
#47°
+
*47°
PCR protocol:
PCR protocol:
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''by Juliette & Terry''
''by Juliette & Terry''
-
From liquid culture made the EDIT !!!!
+
* APRA PJBEI Cl.1
 +
* APRA PJBEI Cl.2
 +
* APRA PJBEI Cl.3
 +
 
 +
From liquid culture made the 6th August.
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Extraction_of_the_Genomic_DNA_from_Bacteria_without_NucleoSpin%C2%AE_Tissue Protocol]
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Extraction_of_the_Genomic_DNA_from_Bacteria_without_NucleoSpin%C2%AE_Tissue Protocol]
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| 1
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 +
 +
==Human Practices==
 +
===Art & Design===
 +
''by Terry''
 +
 +
Our lemon will be made of agar gel. We first thought of buying silicone mold.
 +
But I'll try to make the mold only with agar, cheaper than silicone.
 +
 +
 +
First attempt :
 +
I put about 100ml of hot liquid high density agar ( 35mg/ml ) in a becher and then immerged a test piece to mold.
 +
I let the whole thing cool down in a refrigerator for 2 hours.
 +
Then, I unmold the test piece by cutting accurately around the mold. The mold is placed back in the becher ( without the test piece ), pierced delicately to the cavity and filled up with regular liquid agar ( 20mg/ml ).
 +
Cooled down again in a refrigerator for 2 hours.
 +
 +
BEWARE : The regular agar ( 20mg/ml )is heated to make it liquid but you want to put it in the mold. Too hot, and you will melt the mold. Too cold, and your agar will turn back solid.
 +
 +
Second attempt :
 +
Same protocol than the first attempt, but with higher density agar ( 50mg/ml ).
 +
I let the mold with the test piece in it at refrigerator for the night.
 +
 +
 +
Result : Opening of the first mold, the second one is not cut yet.
 +
 +
[[File:Photo agar test 002.jpg|750px|center]]
 +
 +
From the left to the right :
 +
*agar mold (35mg/ml) cut in two parts
 +
*agar test piece extracted
 +
*original test piece.
 +
*agar mold (50mg/ml) still intact.
 +
==Photo of the Day==
 +
[[File:Paris Saclay 7_august.jpg|600px|center]]
'''Members present:'''
'''Members present:'''

Latest revision as of 14:21, 14 October 2014

Contents

Thursday 7th August

Lab work

Salicylate Inducible Suppressing System

DNA purification gel agarose

by Fabio

Once the segregate process made yesterday by electrophoresis, the DNA correspondent to the core of BBa_K1372000 was purified from the gel agarose.

Segregate Process Protocol

Ligation

by Fabio

The final step to have a BioBrick Assembly is the Ligation reaction.

TODO: illustration of the process

  • BioBrick BBa_K1372000 (Salicylate promoter + NahR + RNA suppressor) as Part A
  • BioBrick BBa_B0015 (Terminator) as Part B

BioBrick Assembly - Ligation Protocol

Lemon Sent

PCR of pCola plasmid (Geranyol Synthase)

by Melanie

we use the same protocol as yesterday but we have done 8 tubes to apply a gradient in the thermocycleur during the third step:

  • 57°
  • 56.3°
  • 55.1°
  • 53.3°
  • 51°
  • 49.3°
  • 47.9°
  • 47°

PCR protocol: 98° --> 2min

5 PCR cycle:

time 10sec 20sec 45sec
temperature 98° (depending on the tube - see the gradient) 72°

25 PCR cycle

time 10sec 20sec 45sec
temperature 98° 72° 72°

and last step: 72° during 10min

but we don't have any results

Test the odor of e.coli with pJBEI6409 plasmide

'by melanie' Transformation of competent cells 5mg1655) by electroporation (pJBEI6409) [1] We do some stock

Extraction of the Genomic DNA

by Juliette & Terry

  • APRA PJBEI Cl.1
  • APRA PJBEI Cl.2
  • APRA PJBEI Cl.3

From liquid culture made the 6th August.

Protocol

Polymerase chain reaction

by Sean & Pierre


for this PCR, five tubes of each of the following Bio-bricks were prepared.

BBa_K517003

component volume
H2O 35.5μl
Phusion buffer 5X 10μl
dNTPs 2μl
iPS68bis 1μl
iPS69 1μl
DNA 1μl
Phusion enzyme 0.5μl


BBa_K762100


component volume
H2O 35.5μl
Phusion buffer 5X 10μl
dNTPs 2μl
iPS66 1μl
iPS67 1μl
DNA 1μl
Phusion enzyme 0.5μl

Tubes were placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

98°C

1 min

1

Denaturation 98°C 15 s 25 - 30
Annealing 52°C 25 s 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1

Human Practices

Art & Design

by Terry

Our lemon will be made of agar gel. We first thought of buying silicone mold. But I'll try to make the mold only with agar, cheaper than silicone.


First attempt : I put about 100ml of hot liquid high density agar ( 35mg/ml ) in a becher and then immerged a test piece to mold. I let the whole thing cool down in a refrigerator for 2 hours. Then, I unmold the test piece by cutting accurately around the mold. The mold is placed back in the becher ( without the test piece ), pierced delicately to the cavity and filled up with regular liquid agar ( 20mg/ml ). Cooled down again in a refrigerator for 2 hours.

BEWARE : The regular agar ( 20mg/ml )is heated to make it liquid but you want to put it in the mold. Too hot, and you will melt the mold. Too cold, and your agar will turn back solid.

Second attempt : Same protocol than the first attempt, but with higher density agar ( 50mg/ml ). I let the mold with the test piece in it at refrigerator for the night.


Result : Opening of the first mold, the second one is not cut yet.

Photo agar test 002.jpg

From the left to the right :

  • agar mold (35mg/ml) cut in two parts
  • agar test piece extracted
  • original test piece.
  • agar mold (50mg/ml) still intact.


Photo of the Day

Paris Saclay 7 august.jpg

Members present:

  • Instructors and advisors: Alice.
  • Students: Eugene, Fabio, Hoang Vu, Juliette, Melanie, Pierre, Romain, Sean and Terry.

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