Difference between revisions of "Team:Paris Saclay/Notebook/July/7"

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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
=Thursday 7<sup>th</sup> July=
 
=Thursday 7<sup>th</sup> July=
−
==Lab work==
+
===Lab work===
 
''By Caroline and Léa''
 
''By Caroline and Léa''
  
−
1L of agarose gel TAE 0,5X agarose 0,8% was prepared following the usual [[Team:Paris_Saclay/Experiments#DNA_electrophoresis_on_agarose_gel|protocol]]
+
1L of agarose TAE gel 0.5X agarose 0.8% was prepared following the usual [[Team:Paris_Saclay/Experiments#DNA_electrophoresis_on_agarose_gel|protocol]]
−
 
+
  
 
===Bringing DNA closer===
 
===Bringing DNA closer===
−
====CAS9 q5 high fidelity PCR ====
+
====CAS9 Q5 high fidelity PCR ====
 
''By Léa''
 
''By Léa''
  
−
Primers IPS 134 and IPS 135 were used to amplify TD-CAS9, NM-cas9 and ST1-cas9. Primers SP1 (IPS 134 + IPS 136) and SP2 (IPS 137 + IPS 135)  were used for DS-SPCasN- .
+
Primers IPS 134 and IPS 135 were used to amplify DS-TDcasN-, DS-NMcasN- and DS-ST1casN-. Primers SP1 (IPS 134 + IPS 136) and SP2 (IPS 137 + IPS 135)  were used for DS-SPCasN- .
 
Primers were diluted in order to get a 100µM final concentration.
 
Primers were diluted in order to get a 100µM final concentration.
−
The usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] for polymerase q5 PCR was followed, with a Tm=65°c.
+
The usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] for polymerase Q5 PCR was followed, with a Tm=65°c.
  
−
====PZA21 q5 high fidelity PCR ====
+
====PZA21 Q5 high fidelity PCR ====
 
‘’By Naiane’’
 
‘’By Naiane’’
  
−
PZA21 plasmids were amplified with primers:
+
PZA21 plasmids were amplified with the primers:
 
* Ter_SPdcas_R and Link-SPdcas_R
 
* Ter_SPdcas_R and Link-SPdcas_R
 
* Link-TDdcas_F and Ter_TDdcas_R
 
* Link-TDdcas_F and Ter_TDdcas_R
Line 24: Line 23:
  
 
Primers stocks (100µM) were diluted as follows:
 
Primers stocks (100µM) were diluted as follows:
−
* Add 280µL of H2O nuclease free to Ter_SPdcas_R and 558 µL to Link-SPdcas_R
+
* Add 280µL of H2O nuclease free to Ter_SPdcas_R and 558 µL to Link-SPdcas_R
−
* Add 599µL of H2O nuclease free to Link-TDdcas_F and 219µL to Ter_TDdcas_R
+
* Add 599µL of H2O nuclease free to Link-TDdcas_F and 219µL to Ter_TDdcas_R
−
* Add 191µL of H2O nuclease free to Ptet F and 315µL to Ptet R.
+
* Add 191µL of H2O nuclease free to Ptet F and 315µL to Ptet R.
  
  
 +
[[File:T--Paris_Saclay--160707_bringingDNAcloser_échelle.jpg|400px|thumb|right|Migration of NM, TD, ST1, ST2, SP, TD, pTet]]
  
 +
====Cas9 PCR====
 +
''By Léa''
  
−
[[File:T--Paris_Saclay--160707_bringingDNAcloser_échelle.jpg|400px|thumb|right|Migration of NM, TD, ST1, ST2, SP, TD, pTet]]
+
Forward primer for addgene Cas9 plasmids (IPS 134) and reverse primer addgene Cas9 plasmids (IPS 135) were used for NM Cas9(DS-NMcasN-), ST1 Cas9 (DS-ST1casN-) and Td Cas9 (DS-TDcasN-).
 +
For SP Cas9 (DS-SPcasN-) we used primers :
 +
* IPS 134 + IPS 136 = SP1
 +
* IPS 137 + IPS 135 = SP2
 +
 
 +
The PCR was conducted following the usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]]
  
 
===Biobrick characterization===
 
===Biobrick characterization===
Line 37: Line 44:
 
''By Laetitia and Charlène''
 
''By Laetitia and Charlène''
  
−
One colony from BL21 was deposited in 4mL of LB and put in incubation at 37°c, 200 rpm ON.
+
One colony from BL21 was incubated in 4mL of LB and put at 37°c, 200 rpm.
  
−
==== Digestion of the plasmid psB1C3 containing the biobrick K1327001====
+
==== Digestion of the plasmid pSB1C3 containing the biobrick K1327001====
 
''By Mathilde and Alice ''
 
''By Mathilde and Alice ''
 
   
 
   
Line 49: Line 56:
  
 
Samples were incubated 5 min at 37°C.  
 
Samples were incubated 5 min at 37°C.  
−
Loading Buffer was added for each solution. Samples were put on an agarose gel for an electrophoresis.
+
Loading Buffer was added for each solution. Samples were put on an agarose gel for electrophoresis.
  
 
[[File:T--Paris_Saclay--160707_characterization_échelle.jpg|200px|thumb|right|Migration of Cl1(K13) and Cl2 (K13)]]
 
[[File:T--Paris_Saclay--160707_characterization_échelle.jpg|200px|thumb|right|Migration of Cl1(K13) and Cl2 (K13)]]
  
−
Clone 1 didn't show any bands.
+
Clone 1 did not show any bands.
−
Clone 2 showed bands at 2000 kb (corresponding to the size of psB1C3) and 1500 kb (corresponding to the size of  biobrick K1327001)
+
Clone 2 showed bands at 2000 kb (corresponding to pSB1C3 size) and 1500 kb (corresponding to biobrick K1327001 size)
  
 
We concluded that clone 2 contain the right insert.
 
We concluded that clone 2 contain the right insert.
  
 
===Visualization===
 
===Visualization===
−
====Electrophoresis migration for gBlocks PCR products ====
+
====PCR products migration====
 
''By Caroline and Léa''
 
''By Caroline and Léa''
  
−
PCR products of pPS16_001, pPS16_002, pPS16_003 pPS16_004, pPS16_005 and pPS16_006 from the [[Team:Paris_Saclay/Notebook/July/6#Visualization│day before]] were each added to 4µL of violet gel loading dye.
+
gBlocks screened the [[Team:Paris_Saclay/Notebook/July/6#Visualization|06/07/2016]] were put on migration.
−
No amplicon was detected.
+
The usual [[Team:Paris_Saclay/Experiments#DNA_electrophoresis_on_agarose_gel|protocol]] was followed.
 +
20µL of each PCR product was added to 4µL of purple loading dye 6X.
  
−
====Results of DH5α|pPS16_004, pPS16_004  clone 6 and pPS16_007 cultures ====
+
====Culture results====
−
Cultures from the [[Team:Paris_Saclay/Notebook/July/6#Visualization│day before]] showed absolutely no blue colony. We suspect a problem with X-Gal IPTG.
+
There was no blue colony observed on xGal/IPTG + ampicillin mediums for [[Team:Paris_Saclay/Notebook/July/6#Visualization|transformation]] with pPS16_004, pPS16_007 and the pPS16_007 clone 6 gBlocks.
  
−
==== DH5α|pPS16_004, pPS16_004  clone 6 and pPS16_007 cultures ====
+
====Culture of bacteria [[Team:Paris_Saclay/Notebook/July/6#Visualization|transformed]] with pPS16_004, pPS16_007 and the pPS16_007 clone 6====
−
‘’ By Laetitia and Charlène’’
+
''By Laetitia and Charlène''
−
In order to solve the X-Gal IPTG issue we had with the previous transformed gBlocks cultures, and to test X-Gal IPTG efficiency, four different mediums were used :
+
−
* New X-Gal, Old X-Gal
+
−
* Old X-Gal, New IPTG
+
−
* New X-Gal, New IPTG
+
−
* Old X-Gal, Old IPTG
+
−
A blue colony was plated on each of those four mediums LB + Ampicillin (50oµL) + X-Gal IPTG (1/1000).
+
−
100 µL of DH5α|pPS16_004, pPS16_004  clone 6 and pPS16_007 were plated again on LB + Ampicillin (50µg/mL) + X-Gal IPTG
+
  
−
====DH5a|pUC19_gBlock Stock of glycerol====
+
Because of the absence of blue colony after incubation on Petri dishes, xGal and IPTG efficency was tested.
−
''By Mathilde and Laetitia ''
+
A Petri dishe was splitted in four parts :
 +
* new xGal, new IPTG
 +
* new xGal, former IPTG
 +
* former xGal, new IPTG
 +
* former xGal, former IPTG
  
−
Transformed cells containing gBlock:
+
A blue colony from the [[Team:Paris_Saclay/Notebook/July/4#Visualization|04/07/2016]] control tube 2 was plated on each of the four part on medium LB + Ampicillin (100µg/mL) + xGal/IPTG (1/100).
−
*1.1 (6clones)
+
−
*1.2 (6clones)
+
−
*2.1(6clones)
+
−
*2.2(6clones)
+
−
*3.1(6clones)
+
−
*3.2(6clones)
+
  
−
For each sample, we made a stock puting 500 of culture and 250 of glycérol (60%)
+
100µL of DH5α|pPS16_004, pPS16_007 or the pPS16_007 clone 6 bacteria were plated again on LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000) medium.
  
 +
====Electrophoresis migration for gBlocks PCR products====
 +
''By Caroline and Léa''
  
 +
PCR products from bacteria transformed with pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005 and pPS16_006 from the [[Team:Paris_Saclay/Notebook/July/6#Visualization|day before]] were each added to 4µL of violet gel loading dye.
 +
But no bands was detected.
  
 +
====Results of DH5α|pPS16_004, pPS16_004 and pPS16_007 clone6 cultures====
 +
Cultures from the [[Team:Paris_Saclay/Notebook/July/6#Visualization|day before]] showed absolutely no blue colony. We suspect a problem with xGal and/or IPTG.
  
 +
====DH5α|pPS16_004, pPS16_004 and pPS16_007 clone6 cultures====
 +
'' By Laetitia and Charlène''
  
 +
In order to solve the xGal/IPTG issues and test xGal/IPTG efficiency, four different mediums were used :
 +
* New X-Gal, Old X-Gal
 +
* Old X-Gal, New IPTG
 +
* New X-Gal, New IPTG
 +
* Old X-Gal, Old IPTG
 +
A blue colony was plated on each of those four mediums LB + Ampicillin (50oµL) + X-Gal IPTG (1/1000).
 +
100 µL of DH5α|pPS16_004, pPS16_004 and pPS16_007 clone6 were plated again on LB + Ampicillin (50µg/mL) + xGal/IPTG
 +
 +
====DH5a|pUC19_gBlock stocks of glycerol====
 +
''By Mathilde and Laetitia ''
  
 +
Transformed cells containing gBlocks:
 +
* 1.1 (6clones)
 +
* 1.2 (6clones)
 +
* 2.1 (6clones)
 +
* 2.2 (6clones)
 +
* 3.1 (6clones)
 +
* 3.2 (6clones)
  
 +
For each sample, stocks were made putting 500µL of culture and 250 of glycerol (60%).
  
−
Then, plasmidic DNA of the remaining Clone 1 and 2 (from the extraction of 06/07/16) were re-concentrated using the following protocol:
+
Then, the remaining plasmid DNA from Clone 1 and 2 (from the 06/07/16 extraction) were re-concentrated using the following protocol:
  
−
*Add 2 volumes of cold ethanol (100%)
+
* Add 2 volumes of cold ethanol (100%)
−
*Add the quivalent of1/10 of the remaining DNA of solution III
+
* Add the equivalent of 1/10 of the remaining DNA of solution III
−
*Put it 30 min at -20°C
+
* Put it 30 min at -20°C
−
*Centrifuge  4 min at1700 rpm
+
* Centrifuge  4 min at1700 rpm
−
*Remove the supernatant
+
* Remove the supernatant
−
*Add 1 mL of ethanol at 70% and inverse
+
* Add 1 mL of ethanol at 70% and inverse
−
*Centrifuge 4 min at 1300 rpm
+
* Centrifuge 4 min at 1300 rpm
−
*Let it dry 1 h
+
* Let it dry 1 h
−
*Dilute with 10 L of sterilized water.
+
* Dilute with 10 L of sterilized water.
−
*Put it at 37°c
+
* Put it at 37°c
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 14:38, 9 October 2016

Thursday 7th July

Lab work

By Caroline and Léa

1L of agarose TAE gel 0.5X agarose 0.8% was prepared following the usual protocol

Bringing DNA closer

CAS9 Q5 high fidelity PCR

By Léa

Primers IPS 134 and IPS 135 were used to amplify DS-TDcasN-, DS-NMcasN- and DS-ST1casN-. Primers SP1 (IPS 134 + IPS 136) and SP2 (IPS 137 + IPS 135) were used for DS-SPCasN- . Primers were diluted in order to get a 100µM final concentration. The usual protocol for polymerase Q5 PCR was followed, with a Tm=65°c.

PZA21 Q5 high fidelity PCR

‘’By Naiane’’

PZA21 plasmids were amplified with the primers:

  • Ter_SPdcas_R and Link-SPdcas_R
  • Link-TDdcas_F and Ter_TDdcas_R
  • Ptet R and Ptet F

Primers stocks (100µM) were diluted as follows:

  • Add 280µL of H2O nuclease free to Ter_SPdcas_R and 558 µL to Link-SPdcas_R
  • Add 599µL of H2O nuclease free to Link-TDdcas_F and 219µL to Ter_TDdcas_R
  • Add 191µL of H2O nuclease free to Ptet F and 315µL to Ptet R.


Migration of NM, TD, ST1, ST2, SP, TD, pTet

Cas9 PCR

By Léa

Forward primer for addgene Cas9 plasmids (IPS 134) and reverse primer addgene Cas9 plasmids (IPS 135) were used for NM Cas9(DS-NMcasN-), ST1 Cas9 (DS-ST1casN-) and Td Cas9 (DS-TDcasN-). For SP Cas9 (DS-SPcasN-) we used primers :

  • IPS 134 + IPS 136 = SP1
  • IPS 137 + IPS 135 = SP2

The PCR was conducted following the usual protocol

Biobrick characterization

Pre-culture of BL21

By Laetitia and Charlène

One colony from BL21 was incubated in 4mL of LB and put at 37°c, 200 rpm.

Digestion of the plasmid pSB1C3 containing the biobrick K1327001

By Mathilde and Alice 

For each sample: 10 μL of DNA 2 μL fast digest buffer 1 μL Pst 1 6 μL H2O

Samples were incubated 5 min at 37°C. Loading Buffer was added for each solution. Samples were put on an agarose gel for electrophoresis.

Migration of Cl1(K13) and Cl2 (K13)

Clone 1 did not show any bands. Clone 2 showed bands at 2000 kb (corresponding to pSB1C3 size) and 1500 kb (corresponding to biobrick K1327001 size)

We concluded that clone 2 contain the right insert.

Visualization

PCR products migration

By Caroline and Léa

gBlocks screened the 06/07/2016 were put on migration. The usual protocol was followed. 20µL of each PCR product was added to 4µL of purple loading dye 6X.

Culture results

There was no blue colony observed on xGal/IPTG + ampicillin mediums for transformation with pPS16_004, pPS16_007 and the pPS16_007 clone 6 gBlocks.

Culture of bacteria transformed with pPS16_004, pPS16_007 and the pPS16_007 clone 6

By Laetitia and Charlène

Because of the absence of blue colony after incubation on Petri dishes, xGal and IPTG efficency was tested. A Petri dishe was splitted in four parts :

  • new xGal, new IPTG
  • new xGal, former IPTG
  • former xGal, new IPTG
  • former xGal, former IPTG

A blue colony from the 04/07/2016 control tube 2 was plated on each of the four part on medium LB + Ampicillin (100µg/mL) + xGal/IPTG (1/100).

100µL of DH5α|pPS16_004, pPS16_007 or the pPS16_007 clone 6 bacteria were plated again on LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000) medium.

Electrophoresis migration for gBlocks PCR products

By Caroline and Léa

PCR products from bacteria transformed with pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005 and pPS16_006 from the day before were each added to 4µL of violet gel loading dye. But no bands was detected.

Results of DH5α|pPS16_004, pPS16_004 and pPS16_007 clone6 cultures

Cultures from the day before showed absolutely no blue colony. We suspect a problem with xGal and/or IPTG.

DH5α|pPS16_004, pPS16_004 and pPS16_007 clone6 cultures

By Laetitia and Charlène

In order to solve the xGal/IPTG issues and test xGal/IPTG efficiency, four different mediums were used :

  • New X-Gal, Old X-Gal
  • Old X-Gal, New IPTG
  • New X-Gal, New IPTG
  • Old X-Gal, Old IPTG

A blue colony was plated on each of those four mediums LB + Ampicillin (50oµL) + X-Gal IPTG (1/1000). 100 µL of DH5α|pPS16_004, pPS16_004 and pPS16_007 clone6 were plated again on LB + Ampicillin (50µg/mL) + xGal/IPTG

DH5a|pUC19_gBlock stocks of glycerol

By Mathilde and Laetitia

Transformed cells containing gBlocks:

  • 1.1 (6clones)
  • 1.2 (6clones)
  • 2.1 (6clones)
  • 2.2 (6clones)
  • 3.1 (6clones)
  • 3.2 (6clones)

For each sample, stocks were made putting 500µL of culture and 250 of glycerol (60%).

Then, the remaining plasmid DNA from Clone 1 and 2 (from the 06/07/16 extraction) were re-concentrated using the following protocol:

  • Add 2 volumes of cold ethanol (100%)
  • Add the equivalent of 1/10 of the remaining DNA of solution III
  • Put it 30 min at -20°C
  • Centrifuge 4 min at1700 rpm
  • Remove the supernatant
  • Add 1 mL of ethanol at 70% and inverse
  • Centrifuge 4 min at 1300 rpm
  • Let it dry 1 h
  • Dilute with 10 L of sterilized water.
  • Put it at 37°c





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