====Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)====
+
====Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6====
−
''By Maxence''
+
''By Maxence & Victor''
−
For that purpose, X clones were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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.
+
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
+
*pPS16_018 (FKBP - GFP 10) clone 6
+
*pPS16_019 (FRB - GFP 11) clone 4
−
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
+
After extraction, 5 µL of each plasmids and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min in order to assess their concentration.
−
* 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:
+
−
{| class="wikitable"
+
−
|-
+
−
!Step
+
−
!Temperature
+
−
!Time
+
−
|-
+
−
|Initial denaturation
+
−
|95°C
+
−
|3 min
+
−
|-
+
−
|rowspan="3"|30 cycles
+
−
|95°C
+
−
|30 sec
+
−
|-
+
−
|48.4°C
+
−
|30 sec
+
−
|-
+
−
|72°C
+
−
|1 min
+
−
|-
+
−
|Final Extension
+
−
|72°C
+
−
|7 min
+
−
|-
+
−
|Hold
+
−
|4°C
+
−
|$\infty$
+
−
|}
+
−
+
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
