Team:Paris/August 27
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{{Paris/Calendar_Links|August 26|August 28}} | {{Paris/Calendar_Links|August 26|August 28}} | ||
- | = | + | =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]] | 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]] | ||
- | 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. | + | <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== | ||
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=> the experiments of Gel Extraction have failed, so we need to repeat the step of digestion. | => the experiments of Gel Extraction have failed, so we need to repeat the step of digestion. | ||
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===Digestion=== | ===Digestion=== | ||
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|EcoRI and XbaI | |EcoRI and XbaI | ||
|} | |} | ||
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==='''Gel Verification'''=== | ==='''Gel Verification'''=== | ||
[[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]] | [[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]] | ||
- | [[Image: | + | [[Image:KR000246.JPG |thumb |Gel Verification of D187 digestion]] |
{|border="1" style="text-align: center" | {|border="1" style="text-align: center" | ||
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|'''Expected size (pb)''' | |'''Expected size (pb)''' | ||
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+ | |2 955 | ||
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- | | | + | |2 940 |
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| colspan="2"| | | colspan="2"| | ||
|- | |- | ||
|'''Measured size (pb)''' | |'''Measured size (pb)''' | ||
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- | |style="background: #cbff7B"| | + | |style="background: #cbff7B"|2 900 |
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- | |style="background: #cbff7B"| | + | |style="background: #cbff7B"|2 900 |
|colspan="2"| | |colspan="2"| | ||
|} | |} | ||
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|2 | |2 | ||
|2 | |2 | ||
+ | |- | ||
+ | |'''number of colonies picked up for screening bis''' | ||
+ | |colspan="3"| | ||
+ | |4 | ||
+ | |4 | ||
+ | | | ||
|} | |} | ||
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==Electrophoresis== | ==Electrophoresis== | ||
- | [[Image:Screening EnvZ.JPG|thumb|]] | + | [[Image:Screening EnvZ.JPG|thumb|Screening of the cloning of EnvZ* - clones 1 to 8]] |
{|border="1" style="text-align:center" | {|border="1" style="text-align:center" | ||
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|'''sample''' | |'''sample''' | ||
|1 kb DNA ladder | |1 kb DNA ladder | ||
- | |positive control | + | |positive<br>control |
- | |negative control | + | |negative<br>control |
- | | | + | |L165.1 |
+ | |L165.2 | ||
+ | |L165.3 | ||
+ | |L165.4 | ||
+ | |L165.5 | ||
+ | |L165.6 | ||
+ | |L165.7 | ||
+ | |L165.8 | ||
|100 bp DNA ladder | |100 bp DNA ladder | ||
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|'''expected size''' | |'''expected size''' | ||
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|- | |- | ||
- | |'''measured size''' | + | |style="background: #ff6d73" |'''measured size''' |
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- | |colspan="8"| | + | |colspan="8"|0,3 kb |
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No correct clone | No correct clone | ||
<br>The 8 other clones were also screened. | <br>The 8 other clones were also screened. | ||
- | + | [[Image:KR000256.JPG|thumb|Screening of the cloning of EnvZ* - clones 9 to 16]] | |
'''PCR''' | '''PCR''' | ||
elongation time: 2 min 30 | elongation time: 2 min 30 | ||
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|'''sample''' | |'''sample''' | ||
|1 kb DNA ladder | |1 kb DNA ladder | ||
- | |positive control | + | |positive<br>control |
- | |negative control | + | |negative<br>control |
- | | | + | |L165.9 |
+ | |L165.10 | ||
+ | |L165.11 | ||
+ | |L165.12 | ||
+ | |L165.13 | ||
+ | |L165.14 | ||
+ | |L165.15 | ||
+ | |L165.16 | ||
|100 bp DNA ladder | |100 bp DNA ladder | ||
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|- | |- | ||
|'''expected size''' | |'''expected size''' | ||
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|- | |- | ||
- | |'''measured size''' | + | |style="background: #ff6d73" |'''measured size''' |
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+ | |colspan="8"|0,3 kb | ||
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|- | |- | ||
|} | |} | ||
<br> | <br> | ||
+ | '''Results''': All the clones analysed were not correct. | ||
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*10 µL of template DNA or 10 µL of EB buffer for th blank | *10 µL of template DNA or 10 µL of EB buffer for th blank | ||
*50 µL of pure water | *50 µL of pure water | ||
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{|border="1" style="text-align:center" | {|border="1" style="text-align:center" | ||
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* 1 µL T4 DNA ligase at 400 000 U/mL concentration | * 1 µL T4 DNA ligase at 400 000 U/mL concentration | ||
* O/N at 16°C | * O/N at 16°C | ||
+ | |||
=Checking mutagenesis FliA= | =Checking mutagenesis FliA= | ||
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For this, i digested mutated FliA and non-mutated FliA with EcoRI and PstI and put in migration the digestion products running on gels.<br> | For this, i digested mutated FliA and non-mutated FliA with EcoRI and PstI and put in migration the digestion products running on gels.<br> | ||
- | Results : No digestion for the mutated sequence --> successful mission ! | + | Results : No digestion for the mutated sequence --> successful mission ! |
Latest revision as of 19:16, 9 September 2008
Construction of pFlhB - mRFP Tripart (LVA+)Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)
DigestionMeasurement of the concentration of D187 purifiedProtocol (it's same that for Miniprep) => the experiments of Gel Extraction have failed, so we need to repeat the step of digestion. Digestion
Gel Verification
Cloning of EnvZ* in pSB1A2Transformation results
PCR screening
Electrophoresis
PCR
elongation time: 2 min 30
Results: All the clones analysed were not correct.
Cloning of OmpR*DigestionDetermination of the concentration of DNAWe used the biophotometer
Name of the digestions
Protocol of digestion
Cleaning of the digestion productsLigationDetermination of the concentration of DNAWe used the biophotometer
Protocol of ligation L171
Checking mutagenesis FliAFor this, i digested mutated FliA and non-mutated FliA with EcoRI and PstI and put in migration the digestion products running on gels. Results : No digestion for the mutated sequence --> successful mission ! |