"
Team:Paris Saclay/Notebook/August/7
From 2014.igem.org
(→Extraction of the Genomic DNA) |
m (→Thursday 7th August) |
||
| (11 intermediate revisions not shown) | |||
| Line 2: | Line 2: | ||
=Thursday 7th August= | =Thursday 7th August= | ||
==Lab work== | ==Lab work== | ||
| - | === | + | ===Salicylate Inducible Suppressing System=== |
| - | ====DNA | + | ====DNA purification gel agarose==== |
| - | '' | + | ''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. | ||
| - | === | + | [https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process Segregate Process Protocol] |
| + | ====Ligation==== | ||
| + | ''by Fabio'' | ||
| - | ==== PCR of pCola plasmid ( | + | 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'' | ''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° | |
| - | + | *56.3° | |
| - | + | *55.1° | |
| - | + | *53.3° | |
| - | + | *51° | |
| - | + | *49.3° | |
| - | + | *47.9° | |
| - | + | *47° | |
PCR protocol: | PCR protocol: | ||
| Line 190: | Line 204: | ||
| 1 | | 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. | ||
| + | |||
| + | [[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.
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.
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.
