Team:Potsdam Bioware/Lab/Labjournal/October
From 2012.igem.org
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
PCR: amplification of EYFP and mcherry with overhang of new RAGE-TMD
Investigators: Sascha
Materials:
- Template: EYFP_Bba_E0030 and nano body-geneart-construct
- Phusion-polymerase
- 10x Phusion buffer HF
- dNTPs (10mM)
- eYFP: fwp:nterm. of yfp to TMD; primerVI: c-terminus of eyfp w
- mcherry: rage-fwp:mcherry/rageTMD; rage:endprimer in mcherry/loxp
- Thermocycler
Methods: 2x master mix of each: eYFP and mcherry
reagent | volume [µL] |
10x Phusion HF buffer | 20 |
dNTPs | 2 |
fwp:nterm. of yfp to TMD; rage-fwp:mcherry/rageTMD | 5 |
primerVI: c-terminus of eyfp w; rage:endprimer in mcherry/loxp | 5 |
template EYFP (10ng/µl); template nanobody-geneart-construct (10ng/µl) | 2 |
Phusion Polymerase | 1,0 |
water | 66 |
Program
step | Temperature [°C] | duration [s] | cycles |
initial denaturation | 98 | 30 | 1 |
denaturation | 98 | 8 | 30 |
annealing | gradient: 64,7°C/71°C for eYFP; 62,0°C/66,1°C for nanobody-geneart-construct | 10 | 30 |
elongation | 72 | 15 | 30 |
final elongation | 72 | 600 | 1 |
cooling | 8 | ∞ | 1 |
Topic: preparative gelelectrophoresis of amplified EYFP and mcherry with RAGE-TMD and RAGE-signalpeptide overhangs
Investigator: Sascha
Materials:
- agarose
- 1xTAE-buffer
- 10xFD Green Buffer
Method:
- 1% agarosegel, 55ml
- 120V
Results:
- amplified eYFP and mcherry showed sufficient bp-size after PCR
Further Tasks:
- gel extraction and concentration measurement
- amplification of scFv-fragment and nanobody/Fc
gel extraction of amplified eYFP and mcherry out of 1% agarosegel
Investigators:Sascha
Results:
[eYFP] =
[mcherry] =
Further Taks:
- extension PCR of scFv and nanobody
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
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Investigator: XXX
Aim: XXX
Materials:
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Method:
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Results:
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Further tasks
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