Latest revision as of 17:01, 9 October 2016

Wednesday 31^th August

Visualization

Colony PCR of 8 clones containing fragment 1 and 2 (dCas9 Nm GFP 10) in pSB1C3

Mahnaz

Transformed cells on 26th august which had been grown overnight were used for colony PCR. For that purpose, 8 clones containing dCas9 NM - GFP 10 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	8 min
30 cycles	95°C	30 sec
	Tm	30 sec
	72°C	t
Final Extension	72°C	10 min
Hold	4°C	infinity

Primers used were:

Matrix	Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers	iPS83 and iPS84
Tm	60.2°C
t	4 min

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 migration

The result shows that Gibson assembly did not work out.

@@ Line 1: / Line 1: @@
 {{Team:Paris_Saclay/notebook_header}}
 = Wednesday 31<sup>th</sup> August=
-==Lab work==
 ===Visualization===
+====Colony PCR of 8 clones containing fragment 1 and 2 (dCas9 Nm GFP 10) in pSB1C3====
+''Mahnaz''
+Transformed cells on 26th august which had been grown overnight were used for colony PCR. For that purpose, 8 clones containing dCas9 NM - GFP 10 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 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:
+{| class="wikitable"
+|-
+!Step
+!Temperature
+!Time
+|-
+|Initial denaturation
+|95°C
+|8 min
+|-
+|rowspan="3"|30 cycles
+|95°C
+|30 sec
+|-
+|Tm
+|30 sec
+|-
+|72°C
+|t
+|-
+|Final Extension
+|72°C
+|10 min
+|-
+|Hold
+|4°C
+|infinity
+|}
+[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+{| class="wikitable"
+|-
+!Matrix
+!Clones containing dCas9 NM - GFP 10 in pSB1C3
+|-
+|Primers
+|iPS83 and iPS84
+|-
+|Tm
+|60.2°C
+|-
+|t
+|4 min
+|}
+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"
+|-
+!PCR products
+!Expected band size (bp)
+|-
+|dCas9 NM - GFP 10 - pSB1C3
+|3688
+|}
+[[File: T--Paris_saclay--3108.extension.png|400px|thumb|center|Result of migration]]
+The result shows that Gibson assembly did not work out.
 {{Team:Paris_Saclay/notebook_footer}}

Difference between revisions of "Team:Paris Saclay/Notebook/August/31"

Latest revision as of 17:01, 9 October 2016

Wednesday 31th August

Visualization

Colony PCR of 8 clones containing fragment 1 and 2 (dCas9 Nm GFP 10) in pSB1C3

Wednesday 31^th August