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

(→‎Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3)
(→‎Wednesday 7th September)
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GEL GEL GEL
 
GEL GEL GEL
  
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====Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 ====
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====Samples preparation for sequencing====
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''By Maxence''
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''by Maxence''
  
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Q5 PCR was performed on plasmids with the following protocol:
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20 µL of plasmides dCas9 ST - GFP 11 (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.
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For each 50μl of reaction, mix the following reagents :
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* 1 µL of plasmid
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* 1 µL of dNTPs (10mM)
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* 1 µL of each primer mix (10µM)
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* 10 µL of Q5 buffer (5X)
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* 0,5 µL of Q5 high fidelity polymerase
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* 35,5 µL of nuclease free water
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Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
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Perform PCR as follow:
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{| class="wikitable"
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|-
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!Step
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!Temperature
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!Time
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|-
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|Initial denaturation
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|98°C
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|30sec
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|-
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|rowspan="3"|30 cycles
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|98°C
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|10sec
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|-
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|Tm
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|20sec
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|-
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|72°C
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|t
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|-
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|Final Extension
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|72°C
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|2min
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|-
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|Hold
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|4°C
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|$\infinity\$
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|}
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
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{| class="wikitable"
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|-
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!Matrix
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!dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8
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!FRB in pJET clones 4 and 9
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!GFP 1.9 in pUC19
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|Primers
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|iPS152 and iPS151
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|iPS149 and iPS150
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|iPS84 and iPS140
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|-
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|Tm
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|57,5°C
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|56,7°C
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|72°C
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|-
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|t
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|1 min 30
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|20 sec
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|30 sec
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|}
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====NanoDrop Measurements====
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''By Maxence''
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{| class="wikitable"
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!Sample
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!Concentration (ng/µL)
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|-
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|PCR fragment GFP 11 clone 6<div id="PCR fragment GFP 11 - pSB1C3 clones 6"></div>
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|187.23
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|PCR fragment GFP 11 clone 8<div id="PCR fragment GFP 11 - pSB1C3 clones 8"></div>
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|156.85
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|-
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|PCR fragment FRB clone 4<div id="PCR fragment FRB clone 4"></div>
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|75.67
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|-
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|PCR fragment FRB clone 9<div id="PCR fragment FRB clone 9"></div>
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|246.41
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|-
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|PCR fragment GFP 1.9<div id="PCR fragment GFP 1.9"></div>
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|22.06
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|}
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====Gel of cleaned up PCR products====
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''By Maxence''
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After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
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PCR products expected were :
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{| class="wikitable"
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|-
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!PCR products
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!Expected band size (bp)
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|-
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|GFP 11- pSB1C3
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|2500
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|FRB
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|374
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|GFP 1.9
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|862
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|}
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[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
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GEL GEL GEL
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Revision as of 13:15, 13 September 2016

Wednesday 7th September

Lab work

Visualization

Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8

By Maxence

Gibson was performed on cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) with the following protocol:

For each 20μl of reaction, mix the following reagents :

  • 0.64 µL of insert
  • 3.68 µL of plasmid
  • 5.67 µL of water
  • 10 µL of buffer

Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL. The PCR was performed as follow : 1 hour at 50°C.

Clean-up of Gibson products

By Maxence

Gibson products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson

By Maxence

Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, or containing control (no mix added when Gibson performed) using the usual protocol.

Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3

Maxence

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 4.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
Tm 30 sec
72°C t
Final Extension 72°C 7 min
Hold 4°C $\infinity\$ 
Primers used were:
Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers iPS83 and iPS84
Tm 63.6°C
t 1 min 30

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
dCas9 NM - GFP 10 - pSB1C3 3688
Result of the migration

GEL GEL GEL

Samples preparation for sequencing

by Maxence

20 µL of plasmides dCas9 ST - GFP 11 (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.