Team:Paris/August 8
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| style="background: #D4E2EF;" | O127 | | style="background: #D4E2EF;" | O127 | ||
| Gene-EnvZ-R | | Gene-EnvZ-R | ||
- | | | + | | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACCCTTCTTTTGTCGTGCCCTGCGCC |
- | | | + | | 60 |
| | | | ||
|- style="background: #dddddd;" | |- style="background: #dddddd;" | ||
| style="background: #D4E2EF;" | O131 | | style="background: #D4E2EF;" | O131 | ||
| Gene-FlhC-R | | Gene-FlhC-R | ||
- | | | + | | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAAACAGCCTGTACTCTCTGTTCATCC |
- | | | + | | 60 |
| | | | ||
|- style="background: #dddddd;" | |- style="background: #dddddd;" | ||
Line 75: | Line 75: | ||
| style="background: #D4E2EF;" | O139 | | style="background: #D4E2EF;" | O139 | ||
| Gene-OmpR-R | | Gene-OmpR-R | ||
- | | | + | | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGGCCCTTTTCTTGCGCAGCGCTTCT |
| 59 | | 59 | ||
| | | | ||
Line 375: | Line 375: | ||
Conclusion | Conclusion | ||
*The bands are not visible on this picture, but with a long exposition time, we managed | *The bands are not visible on this picture, but with a long exposition time, we managed | ||
- | to see the bands | + | to see the bands at their right place. |
*The concentration of DNA is low! | *The concentration of DNA is low! | ||
+ | |||
+ | We will do the ligation [[Team:Paris/August_9|tomorrow]]. | ||
== '''Transformation with promotors of interest'''== | == '''Transformation with promotors of interest'''== | ||
Line 609: | Line 611: | ||
==> '''Conclusion:''' | ==> '''Conclusion:''' | ||
*PCR of pFlgA, pFlgB and pFlhB have succeed, but we always have a problem with pFlhDC probably because of the primers which are not specific. | *PCR of pFlgA, pFlgB and pFlhB have succeed, but we always have a problem with pFlhDC probably because of the primers which are not specific. | ||
+ | |||
+ | |||
+ | =='''Building of the standard measurement plasmid'''== | ||
+ | This morning we MiniPreped E0240 and pSB3K3 which are the two important parts of the standard measurement plasmid. | ||
+ | |||
+ | Before the digestion, we have to determine the DNA concentration of the MiniPreps | ||
+ | |||
+ | ==='''Measurement of DNA concentration'''=== | ||
+ | {| Border="2" | ||
+ | |align="center"|'''Template''' | ||
+ | |align="center"|'''Concentration '''<br>(µg/µL) | ||
+ | |align="center"|'''Ratio DO260/DO280''' | ||
+ | |- | ||
+ | |align="center"|MP 142 <br> pSB3K3 | ||
+ | |align="center"|0.04 | ||
+ | |align="center"|1.76 | ||
+ | |- | ||
+ | |align="center"|MP 143 <br> E0240 | ||
+ | |align="center"|0.16 | ||
+ | |align="center"|1.66 | ||
+ | |} | ||
+ | ==='''Digestion'''=== | ||
+ | ====Protocol==== | ||
+ | {| Border="2" | ||
+ | |align="center"|'''Digestion name''' | ||
+ | |align="center"|'''Template DNA''' | ||
+ | |align="center"|''' Enzymes ''' | ||
+ | |align="center"|'''Volume of DNA''' | ||
+ | |- | ||
+ | |align="center"|D 137 | ||
+ | |align="center"|MP 142 - pSB3K3 | ||
+ | |align="center"|EcoRI-SpeI | ||
+ | |align="center"|25 µL | ||
+ | |- | ||
+ | |align="center"|D 138 | ||
+ | |align="center"|MP 143 - E0240 | ||
+ | |align="center"|EcoRI-SpeI | ||
+ | |align="center"|6.25 µL | ||
+ | |} | ||
+ | |||
+ | * X µL of Template DNA | ||
+ | * Buffer (n°2) 10X : 3µL | ||
+ | * BSA 100X : 0.3µL | ||
+ | * Pure water qsp 30 µL | ||
+ | * 1 µL of each enzyme | ||
+ | |||
+ | * Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). | ||
+ | |||
+ | ====Results of the digestion==== | ||
+ | |||
+ | [[Image:KR000131.jpg|thumb|Result of the digestion to build the measurement plasmid]] | ||
+ | |||
+ | '''Electrophoresis settings''' | ||
+ | * Gel : 1 % agar | ||
+ | * 4µL template DNA for D137 | ||
+ | * All the digestion product for D138 because we have to separate two products, the backbone measures 2056 bp and the insert we want to extract is 899 bp long. | ||
+ | * 10µL QuickLoad DNA ladder 1 kb | ||
+ | |||
+ | |||
+ | {| border="1" | ||
+ | |align="center"|'''Name''' | ||
+ | |align="center"|'''Band''' | ||
+ | |align="center"|'''Expected size''' | ||
+ | |align="center"|'''Measured size''' | ||
+ | |- | ||
+ | |align="center"|D 137 | ||
+ | |align="center"|2 | ||
+ | |style="background: #cbff7B"|<center>2727 bp</center> | ||
+ | |align="center"|3000 bp | ||
+ | |- | ||
+ | |align="center"|D 138 | ||
+ | |align="center"|3&4 | ||
+ | |style="background: #cbff7B"|<center>899 bp</center> | ||
+ | |align="center"|~1000 bp | ||
+ | |} | ||
+ | |||
+ | ==='''Gel extraction and DNA purification'''=== | ||
+ | To extract the biobrick E0240 from the gel, we used the standard protocol [[Team:Paris/Notebook/Protocols#Extraction|number 8]].<br> | ||
+ | To purify the DNA we used the standard protocol [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|number 10]] | ||
+ | |||
+ | The ligation will be done [[Team:Paris/August_9|tomorrow]]. | ||
+ | |||
+ | =='''Building an other [[Team:Paris/Parts/Plastest|measurement]] plasmid'''== | ||
+ | ==='''PCR to create the special E0240'''=== | ||
+ | ====Protocol==== | ||
+ | PCR Mix for cloning with Taq polymerase | ||
+ | * 25µL Quick-load Mix | ||
+ | * 1µL Oligo F (10µM) | ||
+ | * 1µL Oligo R (10µM) | ||
+ | * 1µL Template DNA ([[Team:Paris/Notebook/Freezer#Plasmids:_MiniPrep_Products|MP 143]]) | ||
+ | * 23µL pure water | ||
+ | |||
+ | We try to build a measurement tool with and without included RBS. | ||
+ | {| Border="2" | ||
+ | |align="center"|'''PCR name''' | ||
+ | |align="center"|'''Template DNA''' | ||
+ | |align="center"|'''Oligo F''' | ||
+ | |align="center"|'''Oligo R''' | ||
+ | |- | ||
+ | |align="center"|RBS + | ||
+ | |align="center"|[[Team:Paris/Notebook/Freezer#Plasmids:_MiniPrep_Products|MP 143]] | ||
+ | |align="center"|[[Team:Paris/Notebook/Oligo|O 141]] | ||
+ | |align="center"|[[Team:Paris/Notebook/Oligo|O 140]] | ||
+ | |- | ||
+ | |align="center"|RBS - | ||
+ | |align="center"|[[Team:Paris/Notebook/Freezer#Plasmids:_MiniPrep_Products|MP 143]] | ||
+ | |align="center"|[[Team:Paris/Notebook/Oligo|O 142]] | ||
+ | |align="center"|[[Team:Paris/Notebook/Oligo|O 140]] | ||
+ | |- | ||
+ | |align="center"|T | ||
+ | |align="center"|empty | ||
+ | |align="center"|[[Team:Paris/Notebook/Oligo|O 142]] | ||
+ | |align="center"|[[Team:Paris/Notebook/Oligo|O 140]] | ||
+ | |} | ||
+ | |||
+ | '''PCR Program''' | ||
+ | LID 105°C<br> | ||
+ | 1. 95°C 5 min | ||
+ | 2. 95°C 1 min | ||
+ | 3. 60°C 30 sec | ||
+ | 4. 72°C 1 min 30 | ||
+ | 5. go to : 2 rep : 29 | ||
+ | 6. sound : 1 | ||
+ | 7. hold : 10°C | ||
+ | |||
+ | ====Results==== | ||
+ | [[Image:KR000135.jpg|thumb|Results of the PCR]] | ||
+ | '''Electrophoresis settings''' | ||
+ | * Gel : 1 % agar | ||
+ | * 5µL PCR products | ||
+ | * 10µL QuickLoad DNA ladder 1 kb | ||
+ | |||
+ | {| border="1" | ||
+ | |align="center"|'''Name''' | ||
+ | |align="center"|'''Band''' | ||
+ | |align="center"|'''Expected size''' | ||
+ | |align="center"|'''Measured size''' | ||
+ | |- | ||
+ | |align="center"|RBS + | ||
+ | |align="center"|2 | ||
+ | |style="background: #cbff7B"|<center>900 bp</center> | ||
+ | |align="center"|~ 3000 bp <br> ~ 1000 bp (low fluorescence) | ||
+ | |- | ||
+ | |align="center"|RBS - | ||
+ | |align="center"|3 | ||
+ | |style="background: #cbff7B"|<center>881 bp</center> | ||
+ | |align="center"|~ 3000 bp <br> ~ 1000 bp (strong fluo) | ||
+ | |- | ||
+ | |align="center"|T | ||
+ | |align="center"|4 | ||
+ | |style="background: #cbff7B"|<center>nothing</center> | ||
+ | |align="center"|nothing | ||
+ | |} | ||
+ | |||
+ | Conclusion: | ||
+ | - There is DNA that is around 3,000 bp. Actually, it is the template DNA (MP 143), there were too much DNA. | ||
+ | We will have to extract on gel the right piece of DNA. | ||
+ | - RBS - worked better than RBS +. | ||
+ | |||
+ | ====Gel Extraction and DNA purification==== | ||
+ | [[Image:KR000138.jpg|thumb|Results of the PCR]] | ||
+ | '''Electrophoresis settings''' | ||
+ | * Gel : 1 % agar | ||
+ | * All the PCR products (Two bands for each template) | ||
+ | * 10µL QuickLoad DNA ladder 1 kb | ||
+ | |||
+ | Once again we see that the PCR for RBS - worked a lot better than the PCR for RBS +. We will do it again on [[Team:Paris/August_11|monday]]. | ||
+ | |||
+ | To extract RBS - from the gel, we used the standard protocol [[Team:Paris/Notebook/Protocols#Extraction|number 8]].<br> | ||
+ | To purify the DNA we used the standard protocol [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|number 10]] |
Latest revision as of 13:22, 15 August 2008
Minipreps: Plasmid extractionExtraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.
Amplification of Genes of interest (OmpR, EnvZ, FlhDC)We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
PCR amplificationProtocol
For each sample, 1 µl dNTP
PCR verification/AnalysisAfter the PCR :
ladder : 10µl ladder 1 kb
==> Conclusion : we observed the size expected for the PCR products, but not for FlhDC gene.
Blank : 2µl of buffer EB + 98µl of water
Digestion of PCR productsProtocol :
Analysis by electrophoresis
ladder : 10µl ladder 1 kb
==> Conclusions : We validate the digestion of the vector and the insert. Now we are sure, that we don't detect anything for FlhDC genes. The return of the promotersYesterday, we could not see anything on the electrophoresis after the digestion. Today, we will try the digestion again. Protocol
ResultsTo analyze our digestions, we made an electrophoresis.
Conclusion *The bands are not visible on this picture, but with a long exposition time, we managed to see the bands at their right place. *The concentration of DNA is low! We will do the ligation tomorrow. Transformation with promotors of interestResults
PCR Screening of Ligation TransformantsUse of 8 clones of Ligation transformants for PCR screening
Protocol of screening PCR
25µl of Quick Load 2X
10µl of ladder 100 pb
Results of electrophoresis
Building of the standard measurement plasmidThis morning we MiniPreped E0240 and pSB3K3 which are the two important parts of the standard measurement plasmid. Before the digestion, we have to determine the DNA concentration of the MiniPreps Measurement of DNA concentration
DigestionProtocol
Results of the digestionElectrophoresis settings
Gel extraction and DNA purificationTo extract the biobrick E0240 from the gel, we used the standard protocol number 8. The ligation will be done tomorrow. Building an other measurement plasmidPCR to create the special E0240ProtocolPCR Mix for cloning with Taq polymerase
We try to build a measurement tool with and without included RBS.
PCR Program LID 105°C ResultsElectrophoresis settings
Conclusion: - There is DNA that is around 3,000 bp. Actually, it is the template DNA (MP 143), there were too much DNA. We will have to extract on gel the right piece of DNA. - RBS - worked better than RBS +. Gel Extraction and DNA purificationElectrophoresis settings
Once again we see that the PCR for RBS - worked a lot better than the PCR for RBS +. We will do it again on monday. To extract RBS - from the gel, we used the standard protocol number 8. |