Latest revision as of 17:06, 9 October 2016

Friday 16^th September

Visualization

Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)

By Mahnaz

Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th September. For that purpose, 16 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, 5.13 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
	61°C	30 sec
	72°C	30 sec
Final Extension	72°C	7 min
Hold	4°C	$\infinity\$

Primers used were:

Matrix	Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
Primers	iPS83 and iPS84

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)
FKBP - GFP 10 in pSB1C3 (pPS16_019)	757

Result of the migration

Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.

@@ Line 1: / Line 1: @@
+{{Team:Paris_Saclay/notebook_header}}
 = Friday 16<sup>th</sup> September=
-==Lab work==
 ===Visualization===
-====PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations ====
+====Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)====
-''By Maxence''
+''By Mahnaz''
-In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.
+Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th September. For that purpose, 16 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.
-For each 50μl of reaction, mix the following reagents :
+For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
-* 1 µL of matrix
+* 2.5 µL DreamTaq Buffer
-* 1 µL of dNTPs (10mM)
+* 0.5 µL of dNTPs (10mM)
-* 2.5 µL of each primer mix (10µM)
+* 1 µL of each primer mix (10µM)
-* 10 µL of buffer HF (5X)
+* 0.13 μl of DreamTaq Pol
-* 0,5 µL of Phusion polymerase
-* 32.5 µL of nuclease free water
-Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
+PCR was performed as follow:
-Perform PCR as follow:
 {| class="wikitable"
 |-
@@ Line 25: / Line 24: @@
 |-
 |Initial denaturation
-|98°C
+|95°C
-|30sec
+|3 min
 |-
 |rowspan="3"|30 cycles
-|98°C
+|95°C
-|10sec
+|30 sec
 |-
-|72°C
+|61°C
-|30sec
+|30 sec
 |-
 |72°C
-|t
+|30 sec
 |-
 |Final Extension
 |72°C
-|5min
+|7 min
 |-
 |Hold
 |4°C
-|$\infty$
+|$\infinity\$
 |}
- [[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 {| class="wikitable"
 |-
 !Matrix
-!dCas9 ST - GFP 11 clone 8
+!Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
-!dCas9 ST - GFP 11 clone 8
 |-
 |Primers
-|iPS174 and iPS175
+|iPS83 and iPS84
-|iPS173 and iPS176
 |-
-|Tm
+|}
-|72°C
-|72°C
+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 :
+{| class="wikitable"
 |-
-|t
+!PCR products
-|3min
+!Expected band size (bp)
-|50sec
 |-
+|FKBP - GFP 10 in pSB1C3 (pPS16_019)
+|757
 |}
+[[File:T--Paris Saclay--Gel1118.png|400px|thumb|center|Result of the migration]]
+Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.
+{{Team:Paris_Saclay/notebook_footer}}

Difference between revisions of "Team:Paris Saclay/Notebook/September/16"

Latest revision as of 17:06, 9 October 2016

Friday 16th September

Visualization

Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)

Friday 16^th September