From 2014.igem.org

(Difference between revisions)

Latest revision as of 14:21, 14 October 2014

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.

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

Members present:

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

Back to the calendar

@@ Line 2: / Line 2: @@
 =Thursday 7th August=
 ==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.
+<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:
-#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:
@@ Line 191: / Line 205: @@
 |}
+==Human Practices==
-==Human Pratices==
 ===Art & Design===
 ''by Terry''
@@ Line 198: / Line 211: @@
 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 :
@@ Line 209: / Line 223: @@
 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]]
-Result : Opening of the first mold, the second one is kept in the refrigerator for tomorow.
+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:'''

Team:Paris Saclay/Notebook/August/7