Team:Paris Saclay/Notebook/August/13

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
-
='''Notebook : August 8'''=
+
='''Notebook : August 13'''=
=='''Lab work'''==
=='''Lab work'''==
Line 7: Line 7:
==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Obtaining the PSB3K3 backbone plasmid'''====
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
===='''1 - Electrophoresis of digestion of Bba_K1155000 by SpeI and PstI'''====  
+
===='''1 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI'''====  
Nadia
Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel11308.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*Well 1 : 6µL DNA Ladder
+
* Well 1 : 6µL DNA Ladder
-
*Well 2 : 5µL Bba_K1155000 digested by SpeI and PstI+1µl of 6X loading dye
+
* Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI +1µl of 6X loading dye
-
*Gel : 0.8%
+
* Gel : 0.8%
|}
|}
Expected sizes :  
Expected sizes :  
-
*Pfnr : ...
+
* Pndh* : 111bp
-
*PSB1C3 : ...
+
* pSB1C3 : 2070bp
-
 
+
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We can't see any band for Bba_K1155000 digestion. The digestion failed. We will do it again.
+
We can't see any band for BBa_K1155000 digestion. The digestion failed because we used the wrong enzymes. We will do it again with the good ones.
|}
|}
-
=====2 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI=====
+
===='''2 - Extraction of BBa_K1155004, BBa_1155005, BBa_K1155006 from DH5αstrain'''====
 +
 
 +
XiaoJing
 +
 
 +
Protocol : [[Team:Paris_Saclay/extraction|High-copy plasmid extraction]]
 +
 
 +
===='''3 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and by SpeI/EcoRI'''====
Anaïs, Nadia
Anaïs, Nadia
 +
Used quantities :
 +
* BBa_K1155000 :
 +
** Buffer FD : 5µL
 +
** H2O : 39µL
 +
** DNA : 5µL
 +
** SpeI FD : 1µL
-
''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
+
* BBa_K1155000 :
 +
** Buffer FD : 5µL
 +
** H2O : 38µL
 +
** DNA : 5µL
 +
** SpeI FD : 1µL
 +
**EcoRI FD : 1µL
-
Nadia
+
* BBa_K1155004, BBa_K1155005, BBa_K1155006 :
 +
** Buffer FD : 2µL
 +
** H2O : 7µL
 +
** DNA : 10µL
 +
** SpeI FD : 1µL
-
Protocol : [[Team:Paris_Saclay/Protocols/Electroelution|Electroelution]]
+
* BBa_K1155004, BBa_K1155005, BBa_K1155006 :  
 +
** Buffer FD : 2µL
 +
** H2O : 6µL
 +
** DNA : 10µL
 +
** SpeI FD : 1µL
 +
** EcoRI FD : 1µL
-
We let the plasmid precipitate during the night.
+
We incubat the digestion at 37°C during 10 minutes.
-
===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
+
====4 - '''Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and by SpeI/EcoRI'''====
-
=====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
+
Anaïs, Nadia
-
Anaïs
+
-
*Colony counting :
+
{|
-
**Low concentration petri dish : 47 colonies
+
| style="width:350px;border:1px solid black;" |[[File:Psgel21308.jpg]]
-
**High concentration petri dish : 145 colonies
+
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA Ladder
 +
* Well 2 : 5µL BBa_K1155000 digested by SpeI + 1µl of 6X loading dye
 +
* Well 3 : 5µL BBa_K1155000 +1µl of 6X loading dye
 +
* Well 4 : 5µL BBa_K1155000 digested by EcoRI/Spe I + 1µl of 6X loading dye
 +
* Gel : 0.8%
 +
|}
-
*Picking of 25 colonies
+
Expected sizes :
 +
* Pndh* : 111bp
 +
* pSB1C3 : 2070 kb
-
*Preparation of 700µL of Master mix
+
{|
-
**H<sub>2</sub>O : 590µL
+
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
**dNTP : 28µL
+
We lost all our digestion product so we will do it again.
-
**VF2 primer : 3.5µL
+
|}
-
**VR primer : 3.5µL
+
-
**DreamTaq buffer 10x : 70µL
+
-
**DreamTaq enzyme : 5µL
+
-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
+
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel31308.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 0 : 6µL DNA Ladder
 +
* Well 1 : 5µL BBa_K1155006 digested by SpeI + 1µl of 6X loading dye
 +
* Well 2 : 5µL BBa_K1155005 digested by SpeI + 1µl of 6X loading dye
 +
* Well 3 : 5µL BBa_K1155004 digested by SpeI + 1µl of 6X loading dye
 +
* Well 4 : 5µL BBa_K1155006 digested by EcoRI/SpeI + 1µl of 6X loading dye
 +
* Well 5 : 5µL BBa_K1155005 digested by EcoRI/SpeI + 1µl of 6X loading dye
 +
* Well 6 : 5µL BBa_K1155004 digested by EcoRI/SpeI + 1µl of 6X loading dye
 +
* Gel : 0.8%
 +
|}
-
PCR Program :
+
Expected sizes :  
 +
* NarK, Nar G, NirB : 200kb
 +
* pSB1C3 : 2070bp
 +
* NarK in pSB1C3, NarG in pSB1C3, NirB in pSB1C3 : 2270bp
-
[[File:PsPcr808.jpg|400px]]
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost all our double digestion product so we will do it again. We obtain BBa_K1155004, BBa_K1155005, BBa_K1155006 digested by SpeI fragments at the right size. We can purify it.
 +
|}
-
=====2 - Gel electrophoresis of the colony PCR products=====
+
===='''5 - Liquid culture of MG1655Z1 Δfnr::Km'''====
-
Anaïs, Damir
+
 
 +
XiaoJing
 +
 
 +
We picked up one colony in 5mL of LB and 5µL of kanamycine. We do it twice.
 +
 
 +
We incubated our culture at 37°C with agigation at 150rpm.
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtain fragments at the right size.
 +
|}
 +
 
 +
===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3'''====
 +
 
 +
===='''1 - Digestion of BBa_J04450 by EcoRI/PstI'''====
 +
 
 +
Anaïs
 +
 
 +
Used quantities :
 +
 
 +
* Buffer FD: 2µL
 +
* H2O : 6µL
 +
* DNA : 10µL
 +
* EcoRI FD : 1µL
 +
* PstI FD : 1µL
 +
 
 +
We incubated the digestion at 37°C during 10 minutes.
 +
 
 +
===='''2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI'''====
 +
 
 +
Anaïs, Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel41308.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*6µL DNA Ladder
+
* Well 4 : 6µL DNA Ladder
-
*10µL sample per well
+
* Well 7 : 5µL of BBa_J04450 digested by EcoRI/PstI + 1µL of 6X loading dye
-
*Gel : 0.8%
+
* Gel : 0.8%
|}
|}
-
Expected size : 3583bp
+
Expected sizes :  
 +
* pSB3K3 : 2750bp
 +
* GFP : 1069bp
-
Colonies 10, 14, 15 exhibit plasmids with the right length.
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained a fragment at the right size.
 +
|}
 +
 
 +
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
 +
 
 +
===='''Objective : obtaining FNR and BphR2 proteins (Gibson assembly)'''====
 +
 
 +
===='''1 - PCR of FRN Part I, FNR Part II, BphR2 Part I'''====
 +
 
 +
Anaïs
 +
 
 +
Used quantities :
 +
* Bphr2 Part I :
 +
** Oligo 54F : 1µL
 +
** Oligo 55R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA of ''Pseudomonas pseudoalcaligenes'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* FNR Part I :
 +
** Oligo 59F : 1µL
 +
** Oligo 60R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* FNR Part II :
 +
** Oligo 61F : 1µL
 +
** Oligo 62R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
PCR Program :
 +
 
 +
* BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :
 +
 
 +
[[File:PsPCRBphR23007.jpg|400px]]
 +
 
 +
* FNR Part I, FNR Part II, RBS-FNR Part I :
 +
 
 +
[[File:PsPCRFNR3007.jpg|400px]]
 +
 
 +
===='''2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I'''====
-
====3 - PCR product (made the 08/01/2013) purification====
 
Damir
Damir
-
available quantity:
+
{|
-
* FNR Part1 : 10 µl
+
| style="width:350px;border:1px solid black;" |[[File:Psgel51308.jpg]]
-
* FNR Part2 : 19 µl
+
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* RBS FNR Part1 :16.1µl
+
* Well 1 : 5µL FNR Part II + 1µl of 6X loading dye
-
* RBS BphR2 Part1 : 28µl
+
* Well 2 : 5µL BphR2 Part I + 1µl of 6X loading dye
-
* BphR2 Part1 : 16.4 µl
+
* Well 3 : 5µL FNR Part I + 1µl of 6X loading dye
-
* BphR2 Part2 : 18.9 µl
+
* Well 4 : 6µL DNA Ladder
 +
* Gel : 1%
 +
|}
-
Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
+
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel61308.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 5µL FNR Part II + 1µl of 6X loading dye
 +
* Well 2 : 5µL BphR2 Part I + 1µl of 6X loading dye
 +
* Well 3 : 5µL FNR Part I + 1µl of 6X loading dye
 +
* Well 4 : 6µL DNA Ladder
 +
* Gel : 1%
 +
|}
-
<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.
+
Expected sizes :
 +
* FNR Part I : 597 kb
 +
* FNR Part II : 200 kb
 +
* BphR2 Part I : 178 kb
-
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
It's impossible to read the first gel. We do it again.
 +
In the second gel, we obtain FNR Part I and FNR Part II fragments at the right size. We can purify it. We also obtain BphR2 Part I frangment at the right size but it was mix with other DNA frangments. We will try to make a gel purification of it.
 +
|}
 +
===='''3 - Electrophoresis of PCR product : BphR2 Part I'''====
 +
Anaïs, Damir, Nadia
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel71308.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA Ladder
 +
* Well 2 : 40µL of BphR2 Part I + 8µL of 6X loading dye
 +
* Gel : 1%
 +
|}
 +
 +
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
Even if the two lightest stripes are overlapping, aims to do a second gel purification with these two stripes, we did the gel purification . After argumentation, we decided to do the PCR of BphR2 Part I again thanks to new PCR program and new quantities.
 +
|}
 +
 +
===='''4 - PCR of BphR2 Part I'''====
 +
 +
Anaïs
 +
 +
Used quantities :
 +
* Oligo 54F : 1µL
 +
* Oligo 55R : 1µL
 +
* DNA : 2µL
 +
* Buffer Phusion : 10µL
 +
* dNTP : 1µL
 +
* Phusion : 1µL
 +
* H2O : 33.5µL 
 +
 +
PCR Program :
 +
 +
[[File:PsPCRBphR2PI1308.jpg|400px]]
 +
 +
'''5 - Gel purification of PCR product : FNR Part I, FNR Part II, RBS-FNR Part I, BphR2 Part II,
 +
'''RBS-BphR2 Part I''''''
 +
 +
XiaoJing
 +
 +
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
 +
 +
Nanodrop :
 +
* FNR Part I : 152.7ng/µL
 +
* FNR Part II : 137.2ng/µL
 +
* RBS-FNR Part I : 153.7ng/µL
 +
* BphR2 Part II : 129.9ng/µL
 +
* RBS-BphR2 Part I : 158.1ng/µL
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
The purification was good. We will do the Gibson assembly.
 +
|}
 +
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/12|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/14|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:28, 5 October 2013

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Contents

Notebook : August 13

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI

Nadia

Psgel11308.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI +1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Pndh* : 111bp
  • pSB1C3 : 2070bp

We can't see any band for BBa_K1155000 digestion. The digestion failed because we used the wrong enzymes. We will do it again with the good ones.

2 - Extraction of BBa_K1155004, BBa_1155005, BBa_K1155006 from DH5αstrain

XiaoJing

Protocol : High-copy plasmid extraction

3 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and by SpeI/EcoRI

Anaïs, Nadia

Used quantities :

  • BBa_K1155000 :
    • Buffer FD : 5µL
    • H2O : 39µL
    • DNA : 5µL
    • SpeI FD : 1µL
  • BBa_K1155000 :
    • Buffer FD : 5µL
    • H2O : 38µL
    • DNA : 5µL
    • SpeI FD : 1µL
    • EcoRI FD : 1µL
  • BBa_K1155004, BBa_K1155005, BBa_K1155006 :
    • Buffer FD : 2µL
    • H2O : 7µL
    • DNA : 10µL
    • SpeI FD : 1µL
  • BBa_K1155004, BBa_K1155005, BBa_K1155006 :
    • Buffer FD : 2µL
    • H2O : 6µL
    • DNA : 10µL
    • SpeI FD : 1µL
    • EcoRI FD : 1µL

We incubat the digestion at 37°C during 10 minutes.

4 - Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and by SpeI/EcoRI

Anaïs, Nadia

Psgel21308.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL BBa_K1155000 digested by SpeI + 1µl of 6X loading dye
  • Well 3 : 5µL BBa_K1155000 +1µl of 6X loading dye
  • Well 4 : 5µL BBa_K1155000 digested by EcoRI/Spe I + 1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Pndh* : 111bp
  • pSB1C3 : 2070 kb

We lost all our digestion product so we will do it again.

Psgel31308.jpg
  • Well 0 : 6µL DNA Ladder
  • Well 1 : 5µL BBa_K1155006 digested by SpeI + 1µl of 6X loading dye
  • Well 2 : 5µL BBa_K1155005 digested by SpeI + 1µl of 6X loading dye
  • Well 3 : 5µL BBa_K1155004 digested by SpeI + 1µl of 6X loading dye
  • Well 4 : 5µL BBa_K1155006 digested by EcoRI/SpeI + 1µl of 6X loading dye
  • Well 5 : 5µL BBa_K1155005 digested by EcoRI/SpeI + 1µl of 6X loading dye
  • Well 6 : 5µL BBa_K1155004 digested by EcoRI/SpeI + 1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • NarK, Nar G, NirB : 200kb
  • pSB1C3 : 2070bp
  • NarK in pSB1C3, NarG in pSB1C3, NirB in pSB1C3 : 2270bp

We lost all our double digestion product so we will do it again. We obtain BBa_K1155004, BBa_K1155005, BBa_K1155006 digested by SpeI fragments at the right size. We can purify it.

5 - Liquid culture of MG1655Z1 Δfnr::Km

XiaoJing

We picked up one colony in 5mL of LB and 5µL of kanamycine. We do it twice.

We incubated our culture at 37°C with agigation at 150rpm.

We obtain fragments at the right size.

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3

1 - Digestion of BBa_J04450 by EcoRI/PstI

Anaïs

Used quantities :

  • Buffer FD: 2µL
  • H2O : 6µL
  • DNA : 10µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL

We incubated the digestion at 37°C during 10 minutes.

2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI

Anaïs, Nadia

Psgel41308.jpg
  • Well 4 : 6µL DNA Ladder
  • Well 7 : 5µL of BBa_J04450 digested by EcoRI/PstI + 1µL of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • pSB3K3 : 2750bp
  • GFP : 1069bp

We obtained a fragment at the right size.

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins (Gibson assembly)

1 - PCR of FRN Part I, FNR Part II, BphR2 Part I

Anaïs

Used quantities :

  • Bphr2 Part I :
    • Oligo 54F : 1µL
    • Oligo 55R : 1µL
    • Buffer Phusion : 10µL
    • DNA of Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part I :
    • Oligo 59F : 1µL
    • Oligo 60R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part II :
    • Oligo 61F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL

PCR Program :

  • BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

PsPCRBphR23007.jpg

  • FNR Part I, FNR Part II, RBS-FNR Part I :

PsPCRFNR3007.jpg

2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I

Damir

Psgel51308.jpg
  • Well 1 : 5µL FNR Part II + 1µl of 6X loading dye
  • Well 2 : 5µL BphR2 Part I + 1µl of 6X loading dye
  • Well 3 : 5µL FNR Part I + 1µl of 6X loading dye
  • Well 4 : 6µL DNA Ladder
  • Gel : 1%
Psgel61308.jpg
  • Well 1 : 5µL FNR Part II + 1µl of 6X loading dye
  • Well 2 : 5µL BphR2 Part I + 1µl of 6X loading dye
  • Well 3 : 5µL FNR Part I + 1µl of 6X loading dye
  • Well 4 : 6µL DNA Ladder
  • Gel : 1%

Expected sizes :

  • FNR Part I : 597 kb
  • FNR Part II : 200 kb
  • BphR2 Part I : 178 kb

It's impossible to read the first gel. We do it again. In the second gel, we obtain FNR Part I and FNR Part II fragments at the right size. We can purify it. We also obtain BphR2 Part I frangment at the right size but it was mix with other DNA frangments. We will try to make a gel purification of it.

3 - Electrophoresis of PCR product : BphR2 Part I

Anaïs, Damir, Nadia

Psgel71308.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 40µL of BphR2 Part I + 8µL of 6X loading dye
  • Gel : 1%

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Even if the two lightest stripes are overlapping, aims to do a second gel purification with these two stripes, we did the gel purification . After argumentation, we decided to do the PCR of BphR2 Part I again thanks to new PCR program and new quantities.

4 - PCR of BphR2 Part I

Anaïs

Used quantities :

  • Oligo 54F : 1µL
  • Oligo 55R : 1µL
  • DNA : 2µL
  • Buffer Phusion : 10µL
  • dNTP : 1µL
  • Phusion : 1µL
  • H2O : 33.5µL

PCR Program :

PsPCRBphR2PI1308.jpg

5 - Gel purification of PCR product : FNR Part I, FNR Part II, RBS-FNR Part I, BphR2 Part II, RBS-BphR2 Part I'

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • FNR Part I : 152.7ng/µL
  • FNR Part II : 137.2ng/µL
  • RBS-FNR Part I : 153.7ng/µL
  • BphR2 Part II : 129.9ng/µL
  • RBS-BphR2 Part I : 158.1ng/µL

The purification was good. We will do the Gibson assembly.


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