Difference between revisions of "Team:Paris Saclay/Notebook/August/18"

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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
= Thursday 18<sup>th</sup> August=
 
= Thursday 18<sup>th</sup> August=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
−
====gBlocks 3 digestion with Eco47III====
+
====Fragment 3 digestion with Eco47III====
 
''By Alice''
 
''By Alice''
 +
 +
gBlock 3 were digested with eco47III enzyme following [[Team:Paris_Saclay/Experiments#PlasmidDigestion|this protocol]]. The mix was incubated 2 hours at 37°C. Then eco47III was inactivated during 20 min at 60°C. Then digestion products were purified following [[Team:Paris_Saclay/Experiments#Purification|this protocol]].
 +
 +
====Digested fragment 3 ligation with fragment 4====
 +
''By Alice''
 +
 +
Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mixed in a tube. The mix was incubated 1 hour at room temperature. Then ligation products were purified following [[Team:Paris_Saclay/Experiments#Purification|this protocol]].
 +
 +
====Q5 PCR on fragment 3-4 and Q5 joining PCR on with fragment 3 and fragment 4====
 +
''By Alice''
 +
 +
A Q5 PCR was performed to amplify fragment 3-4 following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]]. The joining of fragments 3 and 4 were done in two different ways. In a first tube we amplified products of fragment 3 and 4 ligation. In a second tube, we did a joining PCR to amplify together fragment 3 and 4 since the undigested fragment 3 has 40 bp overlap with fragment 4. [[Team:Paris_Saclay/Experiments#primers|iPS83 and iPS84]] primers were used. Annealing temprature was 72°C. Initial elongation lasted 30 sec and elongation step lasted 2 min.
 +
 +
PCR products expected were :
 +
 +
{| class="wikitable"
 +
|-
 +
!Fragment
 +
!expected band size (bp)
 +
|-
 +
|3-4
 +
|3919
 +
|}
 +
 +
[[File:T--Paris Saclay--20160818 3-4 fragments joining.JPG|500px|thumb|right|Gel Electrophoresis of PCR products of joining PCR and ligation of 3 and 4 fragments.]]
 +
 +
 +
 +
The bands have not the good size so a new ligation/joining PCR will be done.
 +
 +
====PCR Clean-up of gel bands 1.2 (gBlock) and 4 (ligation)====
 +
''By Naiane''
 +
 +
The bands 1.2 and 4 excised yesterday were used to continue the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 +
====NanoDrop Measurements====
 +
''By Naiane''
 +
 +
{| class="wikitable"
 +
!Sample
 +
!Concentration (ng/µL)
 +
|-
 +
|PCR fragment 2<div id="PCR fragment 2"></div>
 +
|12
 +
|-
 +
|PCR fragment 3<div id="PCR fragment 3"></div>
 +
|14.95
 +
|-
 +
|PCR fragment 4<div id="PCR fragment 4"></div>
 +
|11.36
 +
|-
 +
|PCR fragment 1.2<div id="PCR fragment 1.2"></div>
 +
|15.53
 +
|-
 +
|GFP 1-9 (clone 2)<div id="GFP 1-9 (clone 2)"></div>
 +
|103.75
 +
|-
 +
|GFP 1-9 (clone 3)<div id="GFP 1-9 (clone 3)"></div>
 +
|226.19
 +
|-
 +
|}
 +
 +
====Samples preparation for sequencing====
 +
''by Naiane''
 +
 +
20 µL of plasmides pSB1C3 GFP 1-9 (clone 2) and GFP 1-9 (clone 3) were sent to be sequenced. 20 µL of the primers iPS83 (5µM) and iPS84 (5µM) were sent for sequencing.
 +
 +
====DreamTaq PCR on DH5a colonies transformed on the 17/08/2016 with PJet containing 1.2, FRB, FKBP or sgRNA Nm gBlocks====
 +
''By Léa and Terrence''
 +
 +
Dream Taq PCR were performed on 6 colonies of DH5a transformed on the 17/08/2016 with pJET-FRB ligated with the gblocks FRB,FKBP and sgNM.
 +
12 colonies were plated on Petri dishes and 6 were inoculated into tubes containing 3mL of LB agar and ampicillin.
 +
 +
For DH5a pJET / 1.2, 12 colonies were screened, plated and inoculated into tubes.
 +
The PCR was performed using the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|protocol]] with universal primers for pJET (SO501 and SO511).
 +
 +
Extension at 95°C for 5 min and Tm = 52°C, and elongation time of 1 minute.
 +
 +
The PCR were put onto agarose gel for migration.
 +
 +
[[File:T--Paris Saclay--160818 gel1.jpeg|500px|thumb|right|Gel Electrophoresis of PCR product Pjet-sgRNA.
 +
Expected size: gBlock = 362bp + 118bp (number of bp between the two primers on Pjet) = 480.
 +
Clone 5 seems to have been transformed with pJet containing the right insert. It will be extracted, checked and send to sequencing. We will also test clones 1 and 2.
 +
]]
 +
 +
[[File:T--Paris Saclay--160818 gel2.jpeg|500px|thumb|right|Gel Electrophoresis of PCR product Pjet-1.2.
 +
Expected size: gBlock = 960bp + 118bp (number of bp between the two primers on Pjet) = 1078.
 +
Clone 8 and 11 seem to have been transformed with Pjet containing the right insert. It will be extracted, checked and send to sequencing. We will also test clone 1.
 +
]]
 +
 +
[[File:T--Paris Saclay--160818 gel3.jpeg|500px|thumb|right|Gel Electrophoresis of PCR product Pjet-FRB and FKBP.
 +
Expected size:
 +
FRB : gBlock = 374bp + 118bp (number of bp between the two primers on Pjet) = 492.
 +
Clone 2 to 6 seem to have been transformed with Pjet containing the right insert. Clones 3, 4 and 5 will be extracted, checked and send to sequencing.
 +
 +
FKBP :gBlock = 419bp + 118bp (number of bp between the two primers on Pjet) = 537.
 +
Clone 3, 4 and 5 seem to have been transformed with Pjet containing the right insert. It will be extracted, checked and send to sequencing.]]
 +
 +
====Culture of pPS16_016====
 +
''By Léa and Terrence''
 +
 +
5mL of liquid LB containing chloramphenicol were inoculated with pPS16_016 clones 4 or 5 (pSB1C3 containing gBlock GFP1-9).
 +
The cultures were incubated at 37°C, 180rpm ON.
 +
 
===Biobrick characterization===
 
===Biobrick characterization===
 
====B-Galactosidase and luciferase test on transformed BL21====
 
====B-Galactosidase and luciferase test on transformed BL21====
 
''By Charlene and Mathilde''
 
''By Charlene and Mathilde''
  
−
Cultures were spun down for 5min at 1300rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rom at 4°C.
+
Cultures were spun down for 5min at 13000rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc buffer was added and cells were centrifuged for 15min at 13000rpm at 4°C.
  
 
For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine.
 
For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine.
 
For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added.
 
For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added.
 
OD<sub>420nm</sub> was measured.
 
OD<sub>420nm</sub> was measured.
 +
 +
 +
[[File:T--Paris Saclay--20160818 caracterisation K1372001.jpg|800px|thumb|center|Graphic results of bGal/Luciferase tests on BL21 transformed with K1372001 and pcl_TAA or pcl_TAG or pcl_Tq. 100% represents the activity in Tq condition. TAA condition : BL21 transformed with K1372001 and pcl_TAA. TAG condition : BL21 transformed with K1372001 and pcl_TAG. Tq condition : BL21 transformed with K1372001 and pcl_Tq.]]
 +
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 16:51, 9 October 2016

Thursday 18th August

Visualization

Fragment 3 digestion with Eco47III

By Alice

gBlock 3 were digested with eco47III enzyme following this protocol. The mix was incubated 2 hours at 37°C. Then eco47III was inactivated during 20 min at 60°C. Then digestion products were purified following this protocol.

Digested fragment 3 ligation with fragment 4

By Alice

Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mixed in a tube. The mix was incubated 1 hour at room temperature. Then ligation products were purified following this protocol.

Q5 PCR on fragment 3-4 and Q5 joining PCR on with fragment 3 and fragment 4

By Alice

A Q5 PCR was performed to amplify fragment 3-4 following this protocol. The joining of fragments 3 and 4 were done in two different ways. In a first tube we amplified products of fragment 3 and 4 ligation. In a second tube, we did a joining PCR to amplify together fragment 3 and 4 since the undigested fragment 3 has 40 bp overlap with fragment 4. iPS83 and iPS84 primers were used. Annealing temprature was 72°C. Initial elongation lasted 30 sec and elongation step lasted 2 min.

PCR products expected were :

Fragment expected band size (bp)
3-4 3919
Gel Electrophoresis of PCR products of joining PCR and ligation of 3 and 4 fragments.


The bands have not the good size so a new ligation/joining PCR will be done.

PCR Clean-up of gel bands 1.2 (gBlock) and 4 (ligation)

By Naiane

The bands 1.2 and 4 excised yesterday were used to continue the NucleoSpin Gel and PCR Clean-up kit protocol.

NanoDrop Measurements

By Naiane

Sample Concentration (ng/µL)
PCR fragment 2
 12
PCR fragment 3
 14.95
PCR fragment 4
 11.36
PCR fragment 1.2
 15.53
GFP 1-9 (clone 2)
 103.75
GFP 1-9 (clone 3)
 226.19

Samples preparation for sequencing

by Naiane

20 µL of plasmides pSB1C3 GFP 1-9 (clone 2) and GFP 1-9 (clone 3) were sent to be sequenced. 20 µL of the primers iPS83 (5µM) and iPS84 (5µM) were sent for sequencing.

DreamTaq PCR on DH5a colonies transformed on the 17/08/2016 with PJet containing 1.2, FRB, FKBP or sgRNA Nm gBlocks

By Léa and Terrence

Dream Taq PCR were performed on 6 colonies of DH5a transformed on the 17/08/2016 with pJET-FRB ligated with the gblocks FRB,FKBP and sgNM. 12 colonies were plated on Petri dishes and 6 were inoculated into tubes containing 3mL of LB agar and ampicillin.

For DH5a pJET / 1.2, 12 colonies were screened, plated and inoculated into tubes. The PCR was performed using the usual protocol with universal primers for pJET (SO501 and SO511).

Extension at 95°C for 5 min and Tm = 52°C, and elongation time of 1 minute.

The PCR were put onto agarose gel for migration.

Gel Electrophoresis of PCR product Pjet-sgRNA. Expected size: gBlock = 362bp + 118bp (number of bp between the two primers on Pjet) = 480. Clone 5 seems to have been transformed with pJet containing the right insert. It will be extracted, checked and send to sequencing. We will also test clones 1 and 2.
Gel Electrophoresis of PCR product Pjet-1.2. Expected size: gBlock = 960bp + 118bp (number of bp between the two primers on Pjet) = 1078. Clone 8 and 11 seem to have been transformed with Pjet containing the right insert. It will be extracted, checked and send to sequencing. We will also test clone 1.
Gel Electrophoresis of PCR product Pjet-FRB and FKBP. Expected size: FRB : gBlock = 374bp + 118bp (number of bp between the two primers on Pjet) = 492. Clone 2 to 6 seem to have been transformed with Pjet containing the right insert. Clones 3, 4 and 5 will be extracted, checked and send to sequencing. FKBP :gBlock = 419bp + 118bp (number of bp between the two primers on Pjet) = 537. Clone 3, 4 and 5 seem to have been transformed with Pjet containing the right insert. It will be extracted, checked and send to sequencing.

Culture of pPS16_016

By Léa and Terrence

5mL of liquid LB containing chloramphenicol were inoculated with pPS16_016 clones 4 or 5 (pSB1C3 containing gBlock GFP1-9). The cultures were incubated at 37°C, 180rpm ON.

Biobrick characterization

B-Galactosidase and luciferase test on transformed BL21

By Charlene and Mathilde

Cultures were spun down for 5min at 13000rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc buffer was added and cells were centrifuged for 15min at 13000rpm at 4°C.

For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured.


Graphic results of bGal/Luciferase tests on BL21 transformed with K1372001 and pcl_TAA or pcl_TAG or pcl_Tq. 100% represents the activity in Tq condition. TAA condition : BL21 transformed with K1372001 and pcl_TAA. TAG condition : BL21 transformed with K1372001 and pcl_TAG. Tq condition : BL21 transformed with K1372001 and pcl_Tq.





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