Difference between revisions of "Team:Paris Saclay/Notebook/July/8"

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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
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=Tuesday 8<sup>th</sup> July=
+
=Friday 8<sup>th</sup> July=
−
==Lab work==
+
 
 +
===Bringing DNA closer===
 +
 
 +
[[File:T--Paris_Saclay--160708_bringingDNAcloser_échelle.jpg|400px|thumb|right|Migration of SP, SP1, SP2, NM, ST1, TD]]
  
 
===Visualization===
 
===Visualization===
−
====Colony screening PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/June/28#pPS16_004|pPS16_004]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]]====
+
====Colony screening PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/July/4#pPS16_004|pPS16_004]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]]====
 
''By Alice''
 
''By Alice''
 +
<div id="Colony_screening_PCR"></div>
  
−
After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream  the insertion site are chosen. . PCR products expected below :
+
After transformations, only white bacteria were selected (blue/white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers ([[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]]) upstream and downstream  the insertion site are chosen. Annealing temperature was 53°C. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were put on gel and migrated at 100v during 30 min.
 +
 
 +
PCR products expected were :
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Plasmids
 
!Plasmids
−
!Band size (bp)
+
!expected band size (bp)
 
|-
 
|-
 
|pPS16_004
 
|pPS16_004
−
|846
+
|865
 
|-
 
|-
 
|pPS16_007
 
|pPS16_007
−
|744
+
|763
 
|}
 
|}
  
−
No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of steril water.
+
[[File:T--Paris_Saclay--160711_visualization2_échelle.jpg|400px|thumb|right|pPS16_004 (2.2) and pPS16_007 (4.1) Migration]]
 +
 
 +
No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of MilliQ and steril water allowing the reduction of RNAse. The same PCR will be performed on July 11.
 +
 
 +
====Electrophoresis of the screening PCR products====
 +
''By Caroline''
 +
 
 +
Amplification products from the 07/07/2016 screening PCR were put in a 0.8% agarose gel containing BET. The products in solution were mixed with purple loading dye and migrated for 30min.
 +
Some clones showed PCR products with lengths close to those expected.
 +
 
 +
====High fidelity PCR<div id="high_fidelity_PCR"></div>====
 +
''By Caroline''
 +
 
 +
The clones (from [[Team:Paris_Saclay/Notebook/July/5#Transformation_5/07|05/07/2016]]) found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme: Q5. We used a high fidelity enzyme to reduce the error ratio and send to sequencing the PCR products. A PCR was carried out using Q5<sup>®</sup> High-Fidelity 2X Master Mix and following the [[Team:Paris_Saclay/Experiments#Q5PCR|usual protocol]] with pUC19 primers [[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]].
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Plasmids
 +
!Number of clones tested
 +
!Expected band size (bp)
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001]]
 +
|2
 +
|998
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_002|pPS16_002]]
 +
|3
 +
|998
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_003|pPS16_003]]
 +
|2
 +
|1061
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]]
 +
|2
 +
|998
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_006|pPS16_006]]
 +
|3
 +
|998
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]]
 +
|1
 +
|746
 +
|}
 +
 
 +
[[File:T--Paris_Saclay--160708_visualization_échelle_pas_sure.jpg|400px|thumb|right|pPS16_001 (1.1), pPS16_002 (1.2) and pPS16_003 (2.1) Migration]]
 +
 
 +
[[File:T--Paris_Saclay--160708_visualization2_échelle.jpg|400px|thumb|right|pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration]]
 +
 
 +
[[File:T--Paris_Saclay--160708_visualization3_échelle.jpg|400px|thumb|right|pPS16_004 (2.2) and pPS16_007 (4.1) Migration]]
 +
 
 +
 
 +
 
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 14:49, 9 October 2016

Friday 8th July

Bringing DNA closer

Migration of SP, SP1, SP2, NM, ST1, TD

Visualization

Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007

By Alice

After transformations, only white bacteria were selected (blue/white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1151_pheoR and 1152_pheoF) upstream and downstream the insertion site are chosen. Annealing temperature was 53°C. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were put on gel and migrated at 100v during 30 min.

PCR products expected were :

Plasmids expected band size (bp)
pPS16_004 865
pPS16_007 763
pPS16_004 (2.2) and pPS16_007 (4.1) Migration

No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of MilliQ and steril water allowing the reduction of RNAse. The same PCR will be performed on July 11.

Electrophoresis of the screening PCR products

By Caroline

Amplification products from the 07/07/2016 screening PCR were put in a 0.8% agarose gel containing BET. The products in solution were mixed with purple loading dye and migrated for 30min. Some clones showed PCR products with lengths close to those expected.

High fidelity PCR

By Caroline

The clones (from 05/07/2016) found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme: Q5. We used a high fidelity enzyme to reduce the error ratio and send to sequencing the PCR products. A PCR was carried out using Q5® High-Fidelity 2X Master Mix and following the usual protocol with pUC19 primers 1151_pheoR and 1152_pheoF.

Plasmids Number of clones tested Expected band size (bp)
pPS16_001 2 998
pPS16_002 3 998
pPS16_003 2 1061
pPS16_005 2 998
pPS16_006 3 998
pPS16_007 1 746
pPS16_001 (1.1), pPS16_002 (1.2) and pPS16_003 (2.1) Migration
pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration
pPS16_004 (2.2) and pPS16_007 (4.1) Migration






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