Team:Paris/August 26
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{{Paris/Calendar_Links|August 25|August 27}} | {{Paris/Calendar_Links|August 25|August 27}} | ||
- | =PCR= | + | =Extraction of EnvZ* and OmpR* from ''E. coli'' genome= |
- | == | + | ==Name of the PCR== |
+ | {|style="text-align: center;" border="2" | ||
+ | |Name | ||
+ | |What's in ? | ||
+ | |Template DNA | ||
+ | |Oligo F | ||
+ | |Oligo R | ||
+ | |- | ||
+ | |PCR 147 | ||
+ | |EnvZ* | ||
+ | |S 120 | ||
+ | |O 126 | ||
+ | |O 127 | ||
+ | |- | ||
+ | |PCR 148 | ||
+ | |OmpR* | ||
+ | |S 119 | ||
+ | |O 138 | ||
+ | |O 139 | ||
+ | |- | ||
+ | |T 147 | ||
+ | |nothing | ||
+ | |no template | ||
+ | |O 126 | ||
+ | |O 127 | ||
+ | |- | ||
+ | |T 148 | ||
+ | |nothing | ||
+ | |no template | ||
+ | |O 138 | ||
+ | |O 139 | ||
+ | |} | ||
+ | |||
+ | ==Protocol== | ||
+ | |||
+ | '''Protocol''' | ||
+ | *10 µL Phusion HF Buffer 5X | ||
+ | *2.5 µL Oligo F 10 mM | ||
+ | *2.5 µL Oligo R 10 mM | ||
+ | *1 µL dNTP | ||
+ | *1 µL Template DNA | ||
+ | *33 µL H<sub>2</sub>O | ||
+ | |||
+ | '''PCR Programme''' | ||
+ | *98°C 30s Initial denaturation | ||
+ | *''CYCLE 30X'' | ||
+ | *98°C 10s Denaturation | ||
+ | *60°C 30s Annealing | ||
+ | *72°C 45s Elongation | ||
+ | *''END OF CYCLE'' | ||
+ | *72°C 5min Terminal Elongation | ||
+ | |||
+ | ==Resuts== | ||
+ | [[Image:KR000239.jpg|thumb|Results of the cloning of EnvZ* and OmpR*]] | ||
+ | ===Electrophoresis settings=== | ||
+ | *Gel 1% agar | ||
+ | *10µL Ladder 1kb | ||
+ | *10µL Ladder 100bp | ||
+ | *4µL DNA + 2µL Loading Blue | ||
+ | |||
+ | ===Results of the electrophoresis=== | ||
+ | {| style="text-align: center;" border="1" | ||
+ | |- | ||
+ | |'''Name''' | ||
+ | |'''Gene''' | ||
+ | |'''Well''' | ||
+ | |'''Expected size''' | ||
+ | |'''Measured size''' | ||
+ | |- | ||
+ | |style="background: #cbff7B"|PCR 147 | ||
+ | |EnvZ* | ||
+ | |2 | ||
+ | |1412 bp | ||
+ | |1400 bp | ||
+ | |- | ||
+ | |style="background: #cbff7B"|T 147 | ||
+ | |no sample | ||
+ | |3 | ||
+ | |colspan="2"| | ||
+ | |- | ||
+ | |style="background: #cbff7B"|PCR 148 | ||
+ | |OmpR* | ||
+ | |4 | ||
+ | |779 bp | ||
+ | |800 bp | ||
+ | |- | ||
+ | |style="background: #cbff7B"|T 148 | ||
+ | |no sample | ||
+ | |5 | ||
+ | |colspan="2"| | ||
+ | |} | ||
+ | Conclusion : All the PCR worked perfectly well ! | ||
+ | |||
+ | ==Cleaning of the PCR products== | ||
+ | [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|Standard protocol]] | ||
+ | The cleaned PCR products are stored in the freezer overnight. | ||
+ | |||
+ | |||
+ | |||
+ | =PCR Promoters and Genes FlhDC/FliA= | ||
+ | ==PCR Promoters FlhDC and Gene== | ||
- | * pFlhDC (O111-F / O113-R) | + | * PCR 137 = pFlhDC (O111-F / O113-R) |
- | * Gene FlhDC (O134-F / O131-R) | + | * PCR 141 = Gene FlhDC (O134-F / O131-R) |
- | * pFlgB (O102-F / O103-R) | + | * PCR 125 = pFlgB (O102-F / O103-R) |
- | * pFlhB (O108-F / O109-R) | + | * PCR 126 = pFlhB (O108-F / O109-R) |
---- | ---- | ||
Line 34: | Line 134: | ||
Phusion=0,2µL<br> | Phusion=0,2µL<br> | ||
- | == | + | ==PCR Promoters FliA== |
+ | [[Image:Autres_promoters.jpg|thumb|(49-60)PCR promoter FliA & Promoter+Gene]] | ||
* pFliA (rbs) (O145-F / O144-R) | * pFliA (rbs) (O145-F / O144-R) | ||
Line 65: | Line 166: | ||
Phusion=0,5µL<br> | Phusion=0,5µL<br> | ||
- | == | + | ==PCR mutagenesis FliA== |
- | * PCRFliA1 (O143-F / O152-R) | + | * PCRFliA1 (O143-F / O152-R) |
- | * PCRFliA2 (O153-F / O150-R) | + | * PCR 145 = PCRFliA1' (O148-F / O152-R) |
+ | * PCR 146 = PCRFliA2 (O153-F / O150-R) | ||
* PCRFliA3 (O148-F / O150-R) | * PCRFliA3 (O148-F / O150-R) | ||
---- | ---- | ||
+ | [[Image:PCRFliA1.jpg|thumb|(1-24)PCRFliA1]] | ||
+ | [[Image:PCRFliA2.jpg|thumb|(25-48)PCRFliA2]] | ||
+ | [[Image:PCRFliA1'.jpg|thumb|(61-72)PCRFliA1']] | ||
+ | [[Image:PCRFliA3.jpg|thumb|(73-84)PCRFliA3]] | ||
+ | |||
'''Program: PCRFliA1'''<br> | '''Program: PCRFliA1'''<br> | ||
Line 79: | Line 186: | ||
98°C-10"<br> | 98°C-10"<br> | ||
Gradient 66°C +/-6°C - 25''<br> | Gradient 66°C +/-6°C - 25''<br> | ||
+ | 72°C-20"<br> | ||
+ | '''''Elongation :''''' <br> | ||
+ | 72°C-5'<br> | ||
+ | |||
+ | '''Program: PCRFliA1''''<br> | ||
+ | '''''Denaturation :''''' <br> | ||
+ | 98°C-5'<br> | ||
+ | '''''Cycling 1 (30X) :''''' <br> | ||
+ | 98°C-10"<br> | ||
72°C-20"<br> | 72°C-20"<br> | ||
'''''Elongation :''''' <br> | '''''Elongation :''''' <br> | ||
Line 99: | Line 215: | ||
'''''Denaturation :''''' <br> | '''''Denaturation :''''' <br> | ||
98°C-30'<br> | 98°C-30'<br> | ||
- | '''''Cycling 1 ( | + | '''''Cycling 1 (3X) :''''' <br> |
98°C-10"<br> | 98°C-10"<br> | ||
72°C-30"<br> | 72°C-30"<br> | ||
Line 105: | Line 221: | ||
98°C-10"<br> | 98°C-10"<br> | ||
98°C->72°C low decreasing 1.0°C/s<br> | 98°C->72°C low decreasing 1.0°C/s<br> | ||
+ | 72°C-30"<br> | ||
+ | '''''Break - Add Oligo O148/O150'''''<br> | ||
+ | '''''Cycling 3 (20X) :'''''<br> | ||
+ | 98°C-10"<br> | ||
72°C-30"<br> | 72°C-30"<br> | ||
'''''Elongation :''''' <br> | '''''Elongation :''''' <br> | ||
Line 121: | Line 241: | ||
|2-27 | |2-27 | ||
|197 bp | |197 bp | ||
- | |style="background: #cbff7B"| ~ | + | |style="background: #cbff7B"| ~ 150 bp |
+ | |- | ||
+ | |PCRFliA1' | ||
+ | |Upstream part of FliA | ||
+ | |61-72 | ||
+ | |210 bp | ||
+ | |style="background: #cbff7B"| ~ 180 bp | ||
|- | |- | ||
|PCRFliA2 | |PCRFliA2 | ||
|Downstream part of FliA | |Downstream part of FliA | ||
- | |28- | + | |28-53 |
- | | | + | |670 bp |
- | |style="background: #cbff7B"| | + | |style="background: #cbff7B"| ~ 650 bp |
+ | |- | ||
+ | |PCRFliA3 | ||
+ | |Mutated FliA | ||
+ | |73-84 | ||
+ | |800 bp | ||
+ | |style="background: #cbff7B"| ~ 800 bp | ||
|- | |- | ||
|PCR | |PCR | ||
|pFliA+rbs | |pFliA+rbs | ||
- | | | + | |54-56 |
|325 bp | |325 bp | ||
- | |style="background: #cbff7B"| | + | |style="background: #cbff7B"| ~ 350 bp |
|- | |- | ||
|PCR | |PCR | ||
Line 139: | Line 271: | ||
|57-58 | |57-58 | ||
|310 bp | |310 bp | ||
- | |style="background: #cbff7B"| | + | |style="background: #cbff7B"| ~ 320 bp |
|- | |- | ||
|PCR | |PCR | ||
Line 145: | Line 277: | ||
|59-60 | |59-60 | ||
|1100 bp | |1100 bp | ||
- | |style="background: #cbff7B"| | + | |style="background: #cbff7B"| ~ 1000 bp |
|} | |} | ||
+ | |||
=Cloning of EnvZ*= | =Cloning of EnvZ*= | ||
Line 153: | Line 286: | ||
{|border="1" style="text-align:center" | {|border="1" style="text-align:center" | ||
+ | |'''Digestion n°''' | ||
|'''Name''' | |'''Name''' | ||
|'''[DNA] in µg/mL''' | |'''[DNA] in µg/mL''' | ||
|'''A260/A280''' | |'''A260/A280''' | ||
|- | |- | ||
+ | |D159 | ||
|EnvZ* | |EnvZ* | ||
|10 µg/mL | |10 µg/mL | ||
|1.35 | |1.35 | ||
|- | |- | ||
+ | |D116 | ||
|pSB1A2 | |pSB1A2 | ||
|17 µg/mL | |17 µg/mL | ||
Line 168: | Line 304: | ||
==Ligation== | ==Ligation== | ||
+ | |||
+ | {|border="1" style="text-align:center" | ||
+ | | | ||
+ | |'''control''' | ||
+ | |colspan="3"|'''insert / vector mass ratio''' | ||
+ | |- | ||
+ | | | ||
+ | | | ||
+ | |1,8 / 1 | ||
+ | |2,4 / 1 | ||
+ | |3,1 / 1 | ||
+ | |- | ||
+ | |'''D159 EnvZ* (µL)''' | ||
+ | |0 | ||
+ | |6 | ||
+ | |8 | ||
+ | |8 | ||
+ | |- | ||
+ | |'''D116 pSB1A2 (µL)''' | ||
+ | |2 | ||
+ | |2 | ||
+ | |2 | ||
+ | |1,5 | ||
+ | |- | ||
+ | |'''10X T4 ligase (µL)''' | ||
+ | |2 | ||
+ | |2 | ||
+ | |2 | ||
+ | |2 | ||
+ | |- | ||
+ | |'''T4 DNA ligase (µL)''' | ||
+ | |1 | ||
+ | |1 | ||
+ | |1 | ||
+ | |1 | ||
+ | |- | ||
+ | |'''water (µL)''' | ||
+ | |15 | ||
+ | |9 | ||
+ | |7 | ||
+ | |7,5 | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | *5 hours at room temperature | ||
+ | *transformation of TOP10 competent cells with 5 µL of ligation product | ||
+ | *spreading on LB plates + ampicilline and incubation overnight at 37°C | ||
+ | |||
+ | |||
+ | |||
+ | ='''Miniprep and stock glycerolof New Biobrick'''= | ||
+ | |||
+ | *[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]] | ||
+ | *[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol miniprep]] | ||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Miniprep''' | ||
+ | |'''Strain''' | ||
+ | |'''Antiobiotic''' | ||
+ | |'''Name''' | ||
+ | |'''Description''' | ||
+ | |- | ||
+ | |MP101.1 | ||
+ | |S109.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| J23101 | ||
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>constitutive promotor | ||
+ | |- | ||
+ | |MP101.2 | ||
+ | |S109.2 | ||
+ | |- | ||
+ | |MP101.1 | ||
+ | |S109.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| J23101 | ||
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>constitutive promotor | ||
+ | |- | ||
+ | |MP101.2 | ||
+ | |S109.2 | ||
+ | |- | ||
+ | |MP104.1 | ||
+ | |S111.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| R0040 | ||
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>ptetR repressible | ||
+ | |- | ||
+ | |MP104.2 | ||
+ | |S111.2 | ||
+ | |- | ||
+ | |MP104.1 | ||
+ | |S111.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| R0040 | ||
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>ptetR repressible | ||
+ | |- | ||
+ | |MP104.2 | ||
+ | |S111.2 | ||
+ | |- | ||
+ | |MP105.1 | ||
+ | |S112.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| S03154 | ||
+ | |rowspan="2"| [[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>RBS-lasI | ||
+ | |- | ||
+ | |MP105.2 | ||
+ | |S112.2 | ||
+ | |- | ||
+ | |MP105.1 | ||
+ | |S112.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| S03154 | ||
+ | |rowspan="2"| [[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>RBS-lasI | ||
+ | |- | ||
+ | |MP105.2 | ||
+ | |S112.2 | ||
+ | |- | ||
+ | |MP143 | ||
+ | |S157 | ||
+ | |Amp | ||
+ | |E0240 (in pSB1A2) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>RBS GFP dbl term | ||
+ | |- | ||
+ | |MP1151 | ||
+ | |S150 | ||
+ | |Amp | ||
+ | |L122 <br>D107 (BV) - D130 (BI) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>RBS-lasI - ECFP | ||
+ | |- | ||
+ | |MP157 | ||
+ | |S156 | ||
+ | |Kan | ||
+ | |L138 (E0240 in pSB3K3) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>gfp generator | ||
+ | |- | ||
+ | |MP158.1 | ||
+ | |S160.1 | ||
+ | |rowspan="3"|Amp | ||
+ | |rowspan="3"| L141 | ||
+ | |rowspan="3"| [[Image:Part_icon_regulatory.png]]<br>pFlhD | ||
+ | |- | ||
+ | |MP158.2 | ||
+ | |S160.2 | ||
+ | |- | ||
+ | |MP158.3 | ||
+ | |S160.3 | ||
+ | |- | ||
+ | |MP171 | ||
+ | |S171 | ||
+ | |Amp | ||
+ | |L167 <br>D181 (BV) - D184 (BI) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_regulatory.png]]<br>gfp generator - pTet | ||
+ | |} | ||
+ | |||
+ | |||
+ | |||
+ | ='''Construction of pFlhB - mRFP Tripart (LVA+)'''= | ||
+ | |||
+ | Aim : Construction of ''' "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
+ | <br>We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth. | ||
+ | |||
+ | ==Digestion== | ||
+ | ===Digestion=== | ||
+ | [[Team:Paris/Notebook/Protocols#Digestion|Protocol Digestion]] | ||
+ | |||
+ | {| border="1" style="text-align: center" | ||
+ | |'''Name''' | ||
+ | |'''Template DNA''' | ||
+ | |'''Description''' | ||
+ | |'''Vol MP (µl)''' | ||
+ | |'''Vol H2O (µl)''' | ||
+ | |'''Enzymes''' | ||
+ | |- | ||
+ | |D187 | ||
+ | |MP168.1 | ||
+ | | RBS - mRFP - term (FV) | ||
+ | |9.00 | ||
+ | |15.7 | ||
+ | |EcoRI and XbaI | ||
+ | |} | ||
+ | |||
+ | ==='''Gel Verification'''=== | ||
+ | [[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]] | ||
+ | [[Image:KR000246.JPG |thumb |Gel Verification of D187 digestion]] | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Well''' | ||
+ | |1 | ||
+ | |2 | ||
+ | |3 | ||
+ | |4 | ||
+ | |5 | ||
+ | |6 | ||
+ | |- | ||
+ | |'''Sample''' | ||
+ | |100 pb ladder | ||
+ | |MP168.1 | ||
+ | |no sample | ||
+ | |D187 | ||
+ | |colspan="2"|no sample | ||
+ | |- | ||
+ | |'''Expected size (pb)''' | ||
+ | | | ||
+ | |2 955 | ||
+ | | | ||
+ | |2 940 | ||
+ | | colspan="2"| | ||
+ | |- | ||
+ | |'''Measured size (pb)''' | ||
+ | | | ||
+ | |style="background: #cbff7B"|2 900 | ||
+ | | | ||
+ | |style="background: #cbff7B"|2 900 | ||
+ | |colspan="2"| | ||
+ | |} | ||
+ | |||
+ | |||
+ | ='''Screening of the cloning of pFlgA-GFP Generator'''= | ||
+ | |||
+ | ==Electrophoresis== | ||
+ | [[Image:KR000245.JPG|thumb|Screening of L164]] | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''well n°''' | ||
+ | |1 | ||
+ | |2 | ||
+ | |3 | ||
+ | |4 | ||
+ | |5 | ||
+ | |6 | ||
+ | |7 | ||
+ | |8 | ||
+ | |9 | ||
+ | |- | ||
+ | |'''sample''' | ||
+ | |1kb ladder | ||
+ | |MP172.6 | ||
+ | |MP143.1 | ||
+ | |L161.1 | ||
+ | |L161.2 | ||
+ | |L161.3 | ||
+ | |L161.4 | ||
+ | |L161.5 | ||
+ | |L161.6 | ||
+ | |- | ||
+ | |'''expected size (pb)''' | ||
+ | | | ||
+ | |973 | ||
+ | |1176 | ||
+ | |colspan="6"|1 426 | ||
+ | |- | ||
+ | |'''measured size''' | ||
+ | | | ||
+ | |style="background: #cbff7B"|900 | ||
+ | |style="background: #cbff7B"|1 100 | ||
+ | |style="background: #cbff7B"|1 300 | ||
+ | |style="background: #cbff7B"|1 300 | ||
+ | |style="background: #cbff7B"|1 300 | ||
+ | |style="background: #cbff7B"|1 300 | ||
+ | |style="background: #cbff7B"|1 300 | ||
+ | |style="background: #cbff7B"|1 300 | ||
+ | |} | ||
+ | |||
+ | ==Minipreps and glycerol stock== | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Miniprep''' | ||
+ | |'''Glycerol Stock | ||
+ | |'''Ligation''' | ||
+ | |'''Antibotic''' | ||
+ | |'''Name''' | ||
+ | |- | ||
+ | |MP175.1 | ||
+ | |S177.1 | ||
+ | |L164.1 | ||
+ | |Amp | ||
+ | |[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
+ | FlgA-GFP generator | ||
+ | |- | ||
+ | |MP175.2 | ||
+ | |S177.2 | ||
+ | |L164.2 | ||
+ | |Amp | ||
+ | |[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
+ | FlgA-GFP generator | ||
+ | |- | ||
+ | |MP175.4 | ||
+ | |S177.4 | ||
+ | |L164.4 | ||
+ | |Amp | ||
+ | |[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
+ | FlgA-GFP generator | ||
+ | |} | ||
+ | |||
+ | |||
+ | ='''Construction for Synchronisation'''= | ||
+ | =='''Results of the transformation we did [[Team:Paris/August 24 |yesterday]]'''== | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Ligation name''' | ||
+ | |'''Description''' | ||
+ | |'''Antibio''' | ||
+ | |'''Number Colonies observed''' | ||
+ | |'''Fluorescence''' | ||
+ | |'''Comments''' | ||
+ | |- | ||
+ | |colspan="6" |Ligations | ||
+ | |- | ||
+ | |L159 | ||
+ | |D109(FI) - D125(FV)<br>rbs-lasI - double terminator | ||
+ | |Kan | ||
+ | | - | ||
+ | | - | ||
+ | | to do again | ||
+ | |- | ||
+ | |L162 | ||
+ | |D107(BV) - D163(BI)<br>rbs-lasI - gfp generator | ||
+ | |Amp | ||
+ | | - | ||
+ | | - | ||
+ | | to do again | ||
+ | |- | ||
+ | |L162 | ||
+ | |D110(BV) - D163(BI)<br>rbs-TetR - gfp generator | ||
+ | |Amp | ||
+ | | - | ||
+ | | - | ||
+ | | to do again | ||
+ | |- | ||
+ | |colspan="6" |Controls | ||
+ | |- | ||
+ | |TL159 | ||
+ | |D125(FV) | ||
+ | |Kan | ||
+ | | - | ||
+ | | - | ||
+ | |to do again | ||
+ | |- | ||
+ | |TL162 | ||
+ | |D107(BV) | ||
+ | |Amp | ||
+ | | - | ||
+ | | - | ||
+ | |to do again | ||
+ | |- | ||
+ | |TL163 | ||
+ | |D110(BV) | ||
+ | |AMp | ||
+ | | - | ||
+ | | - | ||
+ | |to do again | ||
+ | |- | ||
+ | |Positive Control | ||
+ | |pUC19 | ||
+ | |Amp | ||
+ | | | ||
+ | |No | ||
+ | |OK | ||
+ | |} |
Latest revision as of 17:59, 11 September 2008
Extraction of EnvZ* and OmpR* from E. coli genomeName of the PCR
ProtocolProtocol
PCR Programme
ResutsElectrophoresis settings
Results of the electrophoresis
Conclusion : All the PCR worked perfectly well ! Cleaning of the PCR productsThe cleaned PCR products are stored in the freezer overnight.
PCR Promoters and Genes FlhDC/FliAPCR Promoters FlhDC and Gene
Program: Gradient Vf=20µL PCR Promoters FliA
Program: promoter Vf=50µL PCR mutagenesis FliA
Program: PCRFliA1' Program: PCRFliA2 Program: PCRFliA3
Cloning of EnvZ*Concentration measurement by Biophotometer
Ligation
Miniprep and stock glycerolof New Biobrick
Construction of pFlhB - mRFP Tripart (LVA+)Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)
DigestionDigestion
Gel Verification
Screening of the cloning of pFlgA-GFP GeneratorElectrophoresis
Minipreps and glycerol stock
Construction for SynchronisationResults of the transformation we did yesterday
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