Revision as of 21:33, 8 September 2013

Notebook : August 21

Damir,Xiaojing

Protocol : Colony PCR By using Master mix B

Damir

[[]]	6µL DNA Ladder 10µL sample per well Gel : 0.8%

Expected size : 3583bp

Nguyen

Incubate at 37°C over night with shake 180 rpm.

obtain plasmid 50µL concentration 195ng/µL

Do 3 tubes, Incubate at 37°C for 2 hours

/

Xiaojing

Sample Volume:

Tube 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)

These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.

@@ Line 36: / Line 36: @@
-PCR Program :
-[[File:PsPcr808.jpg|400px]]
 =====2 - Gel electrophoresis of the colony PCR products=====
@@ Line 53: / Line 51: @@
 Expected size : 3583bp
+====3 - Culture ====
+Nguyen
+*BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol
+*strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine
+Incubate at 37°C over night with shake 180 rpm.
+====4 - Extract plasmid from strain BBa_K115500 clone1 by kit ====
+obtain plasmid 50µL concentration 195ng/µL
+=====5 - Digestion of Bba_K1155000 by SpeI =====
+* Digestion by SpeI :
+**Buffer : 2µL
+**SpeI : 2µL
+**Plasmid : 10µL
+**H20 : 6µL
+Do 3 tubes, Incubate at 37°C for 2 hours
+/
-==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
+==='''A - Aerobic/Anaerobic regulation system / B - PCB se nsing system'''===
-===='''1 - Gibson assembly.'''====
+===='''1 - Gibson assembloy.'''====
 Xiaojing
@@ Line 76: / Line 100: @@
-====3 - PCR pbaroduct (made the 08/01/2013) purification====
-Damirr
-available quantity:
-* FNR Part1 : 10 µl
-* FNR Part2 : 19 µl
-* RBS FNR Part1 :16.1µl
-* RBS BphR2 Part1 : 28µl
-* BphR2 Part1 : 16.4 µl
-* BphR2 Part2 : 18.9 µl
-Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
-<span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.