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From 2010.igem.org

Contents 1 Hassan 2 Nina 3 Colony PCR on IgG protease in shipping vector 4 Mini prep on IgG protease and CPP 5 Sequencing CPP TAT N version

Hassan

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[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]

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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 - ∞

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.

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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:

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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