Team:Stockholm/27 July 2010
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[http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins] | [http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins] | ||
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+ | ==Nina== | ||
+ | |||
+ | ==Colony PCR on IgG protease in shipping vector== | ||
+ | |||
+ | I made a colony PCR on the IgG protease that I have inserted into the iGEM shipping vector to verify that the gene is inserted in a correct position. Therefore I used one of the gene's primers and one of the vector's verification primers. The colony numbers are: 1, 2, 3, 4 and 5. | ||
+ | |||
+ | PCR reaction mix: | ||
+ | |||
+ | * 1 µl Morten's polymerase PjuX7 | ||
+ | |||
+ | * 1 µl 10 mM dNTPs | ||
+ | |||
+ | * 3 µl 5 µM forward primer (VF2) | ||
+ | |||
+ | * 3 µl 5 µM revers primer (gene's primer) | ||
+ | |||
+ | * 10 µl buffer 5X | ||
+ | |||
+ | * 1 µl MgCl2 50mM | ||
+ | |||
+ | * 30 µl H2O | ||
+ | |||
+ | * DNA template was one colony | ||
+ | |||
+ | PCR program: | ||
+ | |||
+ | 98°C - 2 min | ||
+ | |||
+ | 31 cycles of: | ||
+ | |||
+ | * 98°C - 10 sec | ||
+ | |||
+ | * 55°C - 15 sec | ||
+ | |||
+ | * 72°C - 45 sec | ||
+ | |||
+ | 72°C - 5 min | ||
+ | |||
+ | 4°C - ∞ | ||
+ | |||
+ | [[Image:Igg-shipping.jpg|250px]] | ||
+ | |||
+ | DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas | ||
+ | |||
+ | Colony number 2 looks good on the gel. This one will be inoculated in LB in order to become minipreped to be shipped to iGEM hq. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | ==Mini prep on IgG protease and CPP== | ||
+ | |||
+ | I prepare a miniprep on the inoculated IgG protease with colony number 2. In addition I miniprep three inoculated samples with vector carrying CPP from colony number 22, 23 and 33. | ||
+ | |||
+ | The method is carried out according to the procedure in protocols. | ||
+ | |||
+ | Measuring concentration with spectrophotometer: | ||
+ | |||
+ | [[Image:Spek.jpg]] | ||
+ | |||
+ | ---- | ||
+ | |||
+ | ==Sequencing CPP TAT N version== | ||
+ | |||
+ | I send three samples of CPP TAT N version for sequencing. Colony numbers are: 22, 23 and 33. | ||
+ | |||
+ | * 15 ul vector | ||
+ | |||
+ | * 1.5 ul 10uM VR2 primer | ||
+ | |||
+ | 22: ASB0045 105 | ||
+ | |||
+ | 23: ASB0045 104 | ||
+ | |||
+ | 33: ASB0045 103 |
Revision as of 23:39, 31 July 2010
Contents |
Hassan
[http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000364016%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&interactive=yes&network_flavor=actions&targetmode=proteins]
[http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins]
Nina
Colony PCR on IgG protease in shipping vector
I made a colony PCR on the IgG protease that I have inserted into the iGEM shipping vector to verify that the gene is inserted in a correct position. Therefore I used one of the gene's primers and one of the vector's verification primers. The colony numbers are: 1, 2, 3, 4 and 5.
PCR reaction mix:
- 1 µl Morten's polymerase PjuX7
- 1 µl 10 mM dNTPs
- 3 µl 5 µM forward primer (VF2)
- 3 µl 5 µM revers primer (gene's primer)
- 10 µl buffer 5X
- 1 µl MgCl2 50mM
- 30 µl H2O
- DNA template was one colony
PCR program:
98°C - 2 min
31 cycles of:
- 98°C - 10 sec
- 55°C - 15 sec
- 72°C - 45 sec
72°C - 5 min
4°C - ∞
DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas
Colony number 2 looks good on the gel. This one will be inoculated in LB in order to become minipreped to be shipped to iGEM hq.
Mini prep on IgG protease and CPP
I prepare a miniprep on the inoculated IgG protease with colony number 2. In addition I miniprep three inoculated samples with vector carrying CPP from colony number 22, 23 and 33.
The method is carried out according to the procedure in protocols.
Measuring concentration with spectrophotometer:
Sequencing CPP TAT N version
I send three samples of CPP TAT N version for sequencing. Colony numbers are: 22, 23 and 33.
- 15 ul vector
- 1.5 ul 10uM VR2 primer
22: ASB0045 105
23: ASB0045 104
33: ASB0045 103