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Notebook : July 11

Summary:

For régulation system:

1.For those transformation products(of yesterday), RBS+LacS+terminator in PSB1C3 plasmid, the liquid culture has been performed for further experiments. The oligopetides for PCR amplification was made bioinformaticly. The transformation for the terminator BBa_B0010 was still fail.
2. The plasmid which contains fnr+RBS+LacZ+terminator and fnr+RBS+AmilCP+terminator was extracted. The restiction digest was performeed for them.

For sensor system:

3. The selection of bacterian colonies was achieved by analyzing PCR product. The baterian selected are BphR1 c5 and c6; BphR2 c3 and c4; BphA1 c5 and c6,c7,c8.

Lab work

A.aero/anaerobic regulation system:

BioBrick RBS+LacZ+terminator in plasmid PSB1C3

Transformation for BBa_I732019 terminator

After ong night culture, we observed 2 tiny colonies on the medium. We continued the experiments by performing another liquid culture at 37°C with ampicillin.

Transformation for BBa_B0010 was always no go

Verification the transformation of BBa_I004450 in PSB3K3 by digestion and eletrophoresis

We performed 2 types of digestion:

Simple digestion:

DNA: 3µl
Buffe:r 3 µl
Enzyme: 1µl
H2O: 23µl
Total: 30µl

Double digestion:

DNA: 5µl
Buffer: 3 µl
Enzyme: 2µl
H2O: 20µl
Total: 30µl

Buffer used:

Ecor I+ PST I -> orange
Xho I -> green
Sac II -> blue
XhoI+Sac II -> green

After the digestion, we performed a eletrophoresis for verification:

Well 1,7 :XhoI+Sac II
Well 2,8 :Sac II
Well 3,9 :Ecor I+ PST I
Well 4,10 :Xho I
Well 5,11 : control no digested
gel 0.8%

Estimed size and observed size:

enzyme	estimed size	observed size
Ecor I+PST I	1069bp and 2750 bp	1000bp and 2700bp
Xho I	2976bp+842bp	4000bp
Sac II	3819bp	4000bp
Xho I+Sac II	843bp,616bp,2367bp	1200bp and 2000bp

We confimed the existence of BBa_I04550 in plasmid PSB3K3.

B.PCBs sensor system:

Construction for BioBrick promoter promoter BphR1, BphR2, BphA1

Colony PCR

From the culture of cloning for promoter BphR1, BphR2, BphA1 which we seeded yesterday, we had chosen 8 colonies in total for further test. And like we did for promoter fnr, we used 8 primers for PCR amplification, they were: BphR1 up/down, BphR2 up/down, BphA1 up/down and Vf/VR.

In order to make clear this large number of PCR tubes, we classified them into 4 lots. they were:
Mix A : promoter BphR1

buffer go ta:(1X) : 5µl
MgCL2: 2µl
dNTP: 1µl
primers(R1_up/VR or VF/R1_down): 0.125µl
DNA: 2µl
Enzyme: 0.25µl
H2O: about 14.5µl
total: 25µl

Mix B : promoter BphR2

buffer go ta:(1X) : 5µl
MgCL2: 2µl
dNTP: 1µl
primers(R2_up/VR or VF/R2_down): 0.125µl
DNA: 2µl
Enzyme: 0.25µl
H2O: about 14.5µl
total: 25µl

Mix C : promoter BphA1

buffer go ta:(1X) : 5µl
MgCL2: 2µl
dNTP: 1µl
primers(A1_up/VR or VF/A1_down): 0.125µl
DNA: 2µl
Enzyme: 0.25µl
H2O: about 14.5µl
total: 25µl

Mix D :

buffer go ta:(1X) : 5µl
MgCL2: 2µl
dNTP: 1µl
primers(VF/VR): 0.125µL
DNA: 2µl
Enzyme: 0.25µl
H2O: about 14.5µl
total: 25µl

PCR program:

Result : can not find images <p>We considered that promoter BphR1 clone 5 and 6, promoter BphR2 clone 3 and 4, promoter BphA1 clone 5 to 8 accords our estimation. We got them in stock.

Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 23

Lab work

B - PBC sensor system

Objective : obtaining Bba_K1155001, Bba_K1155002, BphR2

'1 - Colony PCR of Bba_K1155001, Bba_K1155002 and BphR2 in DH5α

Anaïs, Zhou

Transformation of Bba_K1155001, Bba_K1155002 and BphR2 protein in DH5α of 07/10/13 work. We will do a Colony PCR.

COLONIES REPIQUEE DANS 10µL d'eau

Used quantities :

DNA : 2µL

Mix A : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- BphR1_Up/VR : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix B : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- VF/BphR1_Down : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix C : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- BphR2_Up/VR : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix D : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- VF/BphR2_Down : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix E : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- BphA1_Up/VR : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix F : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- VF/BphA1_Down : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix G : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 125µL
- MgCl2 : 50µL
- dNTP : 25µL
- VF/VR: 3µL for each oligo
- Enzyme : 6.25µL
- H2O : 362.75µL

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@@ Line 135: / Line 135: @@
 *MgCL2: 2µl
 *dNTP: 1µl
-*primers(VF/VR): 0.125µl
+*primers(VF/VR): 0.125µL
 *DNA: 2µl
 *Enzyme: 0.25µl
@@ Line 152: / Line 152: @@
 <br>
+{{Team:Paris_Saclay/incl_fin}}
+{{Team:Paris_Saclay/incl_debut_generique}}
+='''Notebook : August 23'''=
+=='''Lab work'''==
+==='''B - PBC sensor system'''===
+===='''Objective : obtaining Bba_K1155001, Bba_K1155002, BphR2'''====
+===='''1 - Colony PCR of Bba_K1155001, Bba_K1155002 and BphR2 in DH5α''====
+Anaïs, Zhou
+{|
+| style="border:1px solid black;padding:5px;background-color:#DE;" |
+Transformation of Bba_K1155001, Bba_K1155002 and BphR2 protein in DH5α of 07/10/13 work. We will do a Colony PCR.
+|}
+COLONIES REPIQUEE DANS 10µL d'eau
+Used quantities :
+* DNA : 2µL
+* Mix A : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 50µL
+** MgCl2 : 20µL
+** dNTP : 10µL
+** BphR1_Up/VR : 1.25µL for each oligo
+** Enzyme : 2.5µL
+** H2O : 145µL
+* Mix B : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 50µL
+** MgCl2 : 20µL
+** dNTP : 10µL
+** VF/BphR1_Down : 1.25µL for each oligo
+** Enzyme : 2.5µL
+** H2O : 145µL
+* Mix C : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 50µL
+** MgCl2 : 20µL
+** dNTP : 10µL
+** BphR2_Up/VR : 1.25µL for each oligo
+** Enzyme : 2.5µL
+** H2O : 145µL
+* Mix D : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 50µL
+** MgCl2 : 20µL
+** dNTP : 10µL
+** VF/BphR2_Down : 1.25µL for each oligo
+** Enzyme : 2.5µL
+** H2O : 145µL
+* Mix E : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 50µL
+** MgCl2 : 20µL
+** dNTP : 10µL
+** BphA1_Up/VR : 1.25µL for each oligo
+** Enzyme : 2.5µL
+** H2O : 145µL
+* Mix F : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 50µL
+** MgCl2 : 20µL
+** dNTP : 10µL
+** VF/BphA1_Down : 1.25µL for each oligo
+** Enzyme : 2.5µL
+** H2O : 145µL
+* Mix G : (it was divided in 8tubes for 8 colonies different)
+** Buffer Go Taq : 125µL
+** MgCl2 : 50µL
+** dNTP : 25µL
+** VF/VR: 3µL for each oligo
+** Enzyme : 6.25µL
+** H2O : 362.75µL
 {| border="1" align="center"
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Team:Paris Saclay/Notebook/July/11

From 2013.igem.org

Revision as of 23:00, 23 September 2013

Contents

Notebook : July 11

Summary:

Lab work

Notebook : August 23

Lab work

B - PBC sensor system

Objective : obtaining Bba_K1155001, Bba_K1155002, BphR2

'1 - Colony PCR of Bba_K1155001, Bba_K1155002 and BphR2 in DH5α