Revision as of 20:37, 18 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining the NarK or NarG or NirB _RBS-LacZ-term in PSB1C3 and in PSB3K3

1 - Gel electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI

Anaïs, Nadia

IMAGE

Well 1 : 6µL DNA Ladder
Well 2 : 5µL of BBa_K1155006 digested by EcoRI/SpeI
Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI
Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI
Gel : 1%

Expected sizes :

PSB1C33 : 2070kb
NarK, NarG, NirB : 200kb

We obtained fragments of the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes AND ADN ?!

2 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

Protocol : Digestion

SpeI :
- Buffer : 2µL
- SpeI : 2µL
- ADN : 15µL
- H20 : 1µL

EcoRI/SpeI :
- Buffer : 3µL
- SpeI : 2µL
- EcoRI : 2µL
- ADN : 20µL
- H20 : 3µL

Obtaining RBS_LacZ+Term_PSB1C3

1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies

Anaïs

Colony counting :
- Low concentration petri dish : 47 colonies
- High concentration petri dish : 145 colonies

Picking of 25 colonies

Preparation of 700µL of Master mix
- H₂O : 590µL
- dNTP : 28µL
- VF2 primer : 3.5µL
- VR primer : 3.5µL
- DreamTaq buffer 10x : 70µL
- DreamTaq enzyme : 5µL

Protocol : Colony PCR

PCR Program :

2 - Gel electrophoresis of the colony PCR products

Anaïs, Damir

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

Expected size : 3583bp

Colonies 10, 14, 15 exhibit plasmids with the right length.

3 - PCR product (made the 08/01/2013) purification

Damir

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 : kit purification

Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.

@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-===='''Obtaining the NarK or NarG or NirB _RBS-LacZ-term in PSB1C3'''====
+===='''Obtaining the NarK or NarG or NirB _RBS-LacZ-term in PSB1C3 and in PSB3K3'''====
 =====1 - Gel electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI=====
@@ Line 27: / Line 27: @@
 *NarK, NarG, NirB : 200kb
-  We obtained fragments of the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes.
+  We obtained fragments of the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes AND ADN ?!
-=====2 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI=====
+=====2 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI=====
+Anaïs, Nadia
-''2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1''
-Anaïs
-{|
-| style="width:350px;border:1px solid black;" | [[File:PsNBa8_eelution.jpg|350px]]
-| style="width:350px;border:1px solid black;vertical-align:top;" |
-*Well 1 : 6µL DNA Ladder
-*Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
-*Gel : 0.8%
-|}
- Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.
-''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
-Nadia
+Protocol : [[Team:Paris_Saclay/Protocols/Digestion|Digestion]]
-Protocol : [[Team:Paris_Saclay/Protocols/Electroelution|Electroelution]]
+* SpeI :
+**Buffer : 2µL
+**SpeI : 2µL
+**ADN : 15µL
+**H20 : 1µL
- We let the plasmid precipitate during the night.
+*EcoRI/SpeI :
+**Buffer : 3µL
+**SpeI : 2µL
+**EcoRI : 2µL
+**ADN : 20µL
+**H20 : 3µL
 ===='''Obtaining RBS_LacZ+Term_PSB1C3'''====

Team:Paris Saclay/Notebook/August/14

From 2013.igem.org