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 | + | 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 | + | ====CAS9 Q5 high fidelity PCR ==== |
''By Léa'' | ''By Léa'' | ||
| − | Primers IPS 134 and IPS 135 were used to amplify | + | 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 | + | The usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] for polymerase Q5 PCR was followed, with a Tm=65°c. |
| − | ====PZA21 | + | ====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'' | ||
| − | + | 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 | + | One colony from BL21 was incubated in 4mL of LB and put at 37°c, 200 rpm. |
| − | ==== Digestion of the plasmid | + | ==== 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 | + | 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 | + | Clone 1 did not show any bands. |
| − | Clone 2 showed bands at 2000 kb (corresponding to | + | 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=== | ||
| − | ==== | + | ====PCR products migration==== |
''By Caroline and Léa'' | ''By Caroline and Léa'' | ||
| − | PCR | + | gBlocks screened the [[Team:Paris_Saclay/Notebook/July/6#Visualization|06/07/2016]] were put on migration. |
| − | + | 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. | ||
| + | |||
| + | ====Culture results==== | ||
| + | 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. | ||
| + | |||
| + | ====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'' | ||
| + | |||
| + | 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 [[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). | ||
| + | |||
| + | 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 | |
| − | In order to solve the | + | * Old X-Gal, New IPTG |
| − | * New X-Gal, Old X-Gal | + | * New X-Gal, New IPTG |
| − | * Old X-Gal, New IPTG | + | * Old X-Gal, Old 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). | 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 | + | 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 | + | ====DH5a|pUC19_gBlock stocks of glycerol==== |
''By Mathilde and Laetitia '' | ''By Mathilde and Laetitia '' | ||
| − | Transformed cells containing | + | Transformed cells containing gBlocks: |
| − | *1.1 (6clones) | + | * 1.1 (6clones) |
| − | *1.2 (6clones) | + | * 1.2 (6clones) |
| − | *2.1(6clones) | + | * 2.1 (6clones) |
| − | *2.2(6clones) | + | * 2.2 (6clones) |
| − | *3.1(6clones) | + | * 3.1 (6clones) |
| − | *3.2(6clones) | + | * 3.2 (6clones) |
| − | For each sample, | + | 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: | 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 equivalent of 1/10 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
