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Team:Potsdam Bioware/Lab/Labjournal/October
From 2012.igem.org
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<b>Materials and Methods:</b> Geneious <br> | <b>Materials and Methods:</b> Geneious <br> | ||
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| + | <p style="background-color: rgb(238, 221, 130); font-weight: bold;"> Topic: Primer design and ordering for integration of RAGE-transmembrane domain into scFVconstruct and nanobody-geneart construct</p> | ||
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| + | <b>Investigator:</b> Sascha <br> | ||
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| + | <b>Materials and Methods:</b> Geneious <br> | ||
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| + | <b>Results:</b><br> | ||
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| + | * scFv:startprimer-fw | ||
| + | ** GCCTCTAGAGCTAGCATGGCAGC | ||
| + | * rvp:scfv+overhang to TEV/TMD | ||
| + | **CGCCCAAGATTCCCAGGGCCAGGGCGAGGGTGCCGCTTCCGCCGCCACCGCTTCCCCCTTGAAAATATAAATTCTCTCCAGATCCCCGTTT | ||
| + | GATCTCCAGTTCTGTCC | ||
| + | * fwp:nterm. of yfp to TMD | ||
| + | ** CCCTGGGAATCTTGGGCGGCCTGGGGACCGCTGCCCTCTTGATCGGCGTGATCCTGTGGCAGAGAAGGTCTGGAGTGAGCAAGGGCGAG | ||
| + | GAGC | ||
| + | * scFv:endprimer-rv | ||
| + | ** GAGCTGCAGCGGCCG | ||
| + | * rage:startprimer-fw_nano/Fc | ||
| + | ** CTTGAATTCGCGGCCG | ||
| + | * rage-rv:Fc to rageTMD | ||
| + | ** CGCCCAAGATTCCCAGGGCCAGGGCGAGGGTGCCGCTTCCTAATAACTTCGTATAATGTATGCTATACGAAGTTATCCC | ||
| + | * rage-fwp:mcherry/rageTMD | ||
| + | ** CCCTGGGAATCTTGGGCGGCCTGGGGACCGCTGCCCTCTTGATCGGCGTGATCCTGTGGCAGAGAAGGGGCTCCATGGTGTCCAAGG | ||
| + | * rage:endprimer in mcherry/loxp | ||
| + | ** AAGTCACTGCAGCGGCC | ||
| + | <br> | ||
===<p style="background-color: rgb(240, 20, 70);">2012-10-15</p>=== | ===<p style="background-color: rgb(240, 20, 70);">2012-10-15</p>=== | ||
Revision as of 23:29, 25 October 2012
Contents |
AID
2012-10-10
Inoculation of plasmid samples of the 48h retransformation plates (after FACS)
Investigators: Tom
Time: 2012-10-10
Materials:
- LB medium
- Amp stock solution
- plate with cultures:
EGFR-C-AID without NES, with NLS+Kozak sequence+eGFP
(after FACS selection)
Method:
Inoculation of:
5 cultures per plate in 5 ml LB medium + 5µL Amp.
(--> 5 cultures)
Further tasks:
- Miniprep
Antibody
2012-10-12
cell culture
Investigator:Kerstin
Topic: cell-culture
- passaging of CHO w Zeocin
- passaging of HT1080
- passaging of HEK AAV 293
- passaging of HeLa
Topic: planning new primer for integration of new RAGE-transmembrane domain and RAGE-signalpeptide
Investigator: Sascha
Materials and Methods: Geneious
Topic: Primer design and ordering for integration of RAGE-transmembrane domain into scFVconstruct and nanobody-geneart construct
Investigator: Sascha
Materials and Methods: Geneious
Results:
- scFv:startprimer-fw
- GCCTCTAGAGCTAGCATGGCAGC
- rvp:scfv+overhang to TEV/TMD
- CGCCCAAGATTCCCAGGGCCAGGGCGAGGGTGCCGCTTCCGCCGCCACCGCTTCCCCCTTGAAAATATAAATTCTCTCCAGATCCCCGTTT
GATCTCCAGTTCTGTCC
- fwp:nterm. of yfp to TMD
- CCCTGGGAATCTTGGGCGGCCTGGGGACCGCTGCCCTCTTGATCGGCGTGATCCTGTGGCAGAGAAGGTCTGGAGTGAGCAAGGGCGAG
GAGC
- scFv:endprimer-rv
- GAGCTGCAGCGGCCG
- rage:startprimer-fw_nano/Fc
- CTTGAATTCGCGGCCG
- rage-rv:Fc to rageTMD
- CGCCCAAGATTCCCAGGGCCAGGGCGAGGGTGCCGCTTCCTAATAACTTCGTATAATGTATGCTATACGAAGTTATCCC
- rage-fwp:mcherry/rageTMD
- CCCTGGGAATCTTGGGCGGCCTGGGGACCGCTGCCCTCTTGATCGGCGTGATCCTGTGGCAGAGAAGGGGCTCCATGGTGTCCAAGG
- rage:endprimer in mcherry/loxp
- AAGTCACTGCAGCGGCC
2012-10-15
cell culture
Investigator:Kerstin
Topic: cell-culture
- passaging of CHO w Zeocin
- passaging of HT1080
- passaging of HEK AAV 293
- passaging of HeLa
2012-10-17
cell culture
Investigator:Kerstin
Topic: cell-culture
- passaging of CHO w Zeocin
- passaging of HT1080
- passaging of HEK AAV 293
- passaging of HeLa
- passaging of stably transfected Clones 4.1, 4.2, 4.3, 4,4, 29.1, 29.2, 29.3 with 550µg/ml Hygromycin
2012-10-19
cell culture
Investigator:Stefan/Kerstin
Topic: cell-culture
- passaging of CHO w Zeocin
- passaging of HT1080
- passaging of HEK AAV 293
- passaging of HeLa
2012-10-22
cell culture
Investigator:Kerstin
Topic: cell-culture
- passaging of CHO w Zeocin
- passaging of HT1080
- passaging of HEK AAV 293
- passaging of HeLa
- transfection of stably transfected clone 29.1 with Cre Recombinase
- seeding of HT1080 in Ibidi Dish for infection with virus
2012-10-23
cell culture
Investigator:Stefan
Topic: cell-culture
- seeding of CHO and HeLa in Ibidi Dishes for transfection with new Nanobody RAGE construct
- infection with Virus (CFP on surface and YFP as GOI, AAV with Sortase motif)
2012-10-24
cell culture
Investigator:Stefan/Kerstin
Topic: cell-culture
- Transfection of new Nanobody RAGE construct in CHO and HeLa
- seeding of CHO and HeLa in Ibidi Dishes for transfection with new scFv RAGE construct
- passaging of CHO w Zeocin
- passaging of HT1080
- passaging of HEK AAV 293
- passaging of HeLa
2012-10-25
cell culture
Investigator:Stefan/Kerstin
Topic: cell-culture
- transfection of new scFv RAGE construct
- transfection of TAL-AID from cooperation with Freiburg iGEM Team in CHO cells (co-transfection with clone 4)
- purification of Nanobody from supernatant of Cre recombinase transfected cells with magnetic beads
- Western Blot of purified Nanobody
Virus
XXX
XXX
Investigator: XXX
Aim: XXX
Materials:
XXX
Method:
XXX
Results:
XXX
Further tasks
XXX
