Team:Potsdam Bioware/Lab/Labjournal/June

==AID-Group=====

1st Labday 2012-06-09

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Topic: Planing the wildtype AID construct (BBa_K929000)

Investigators: Tom S., Chris, Basia, Rico, Mario, Kevin

'''Aim:''' planing the construction of the wildtype AID in pSB1C3

'''Material:''' Genious

'''Results:'''* pSB1C3 with CMV -> (cut with SpeI and PstI) 2715 bp (CMV insert+backbone) + 18 bp (rest)* pSB1A3 with AID -> (cut with XbaI and PstI) 626 bp (AID insert) + 2061 bp (backbone)* pSB1C3 with hGH -> (cut with XbaI and PstI) 505 bp (hGH insert) + 2053 bp (backbone)[[File:UP12_WildtypAID.JPG|300px|AID_Wirkorte]]
'''Further tasks:'''* practice part

Topic: Planing the modified AID construct (BBa_K929002)

Investigators: Tom S., Chris, Basia, Rico, Mario, Kevin

'''Aim:''' planing the modified AID with Kozak sequence, NLS and without NES, Primer design

'''Material:''' Genious

'''Results:'''
[[File:UP12_superAID.JPG|300px]]* reverse primer without NES
'''Further tasks:'''* design of the forward primer* design of the practice part===

2012-06-24

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Topic: Preparation of overnight culture of E. coli strain XL-1 Blue

Investigators:Basia
Aim:preparation of competent cells of E. coli strain XL-1 Blue
Materials:LB Medium, Tetracycline, XL-1 Blue stock
Method:Competent E. coli -> Standard protocols
Results:
culture grew
Further tasks:
further preparation of competent cells with MgCl2 and CaCl2.

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2012-06-25

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Topic: making competent XL1 Blue E. coli

Investigators:Sascha, Maria, Tarek, Chris
Aim:get competent E. coli Xl1 Blue
Materials:CaCl2, Glycerol, overnight culture
Method:Competent E. coli -> Standard protocols
Results:
* ca. 80 (100 µL) Stocks frozen competent E. coli XL1 Blue* location: competent E. coli Xl1 Blue in -80°C FreezerFurther tasks:
* testing ability for transformation of competent cells===

2012-06-27

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Topic: Picking clones/start overnight culture: AID(BBa_K103001); start overnight culture with pcdna5/ftr

Investigators:Chris, Mario
Aim:Picking clones/start 5 mL culture: 2 * 5 mL, 1*20 mL for Miniprep AID(BBa_K103001) & glycerolstock; 1 * 5 mL (Plasmid Sascha:pcdna5/frt) per competent cell line ("good", "bad" and "AG")
Materials:E. coli colonies from agar-plates (antibiotic: Amp), test tubes+ 5 mL LB+1:1000 AMP, 37 °C shaker
Method:picking clones, inoculate in 5 mL fresh LB-media with Ampicilin (1:1000), incubation over night
Results:
ready for Miniprep: 2*5 mL and 1*20 mL E. coli XL1 with AID(BBa_K103001); 1 * 5 mL (Plasmid Sascha:pcdna5/frt) per competent cell line ("good", "bad" and "AG")* location: incubator 37 °C
Further tasks:
*Preparation of glycerol stocks (one of XL1 with AID (BBa_K103001), one of XL1 with pcdna5/frt)*Miniprep Thursday 28.06.2012===

2012-06-28

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Topic: MiniPrep of AID(BBa_K103001) and pcdna5/frt + preparation of cryostocks of the cells

Investigators:Basia

Aim:Miniprep & glycerol stock AID(BBa_K103001); Miniprep and cryostock of pcdna5/frt per competent cell line ("good", "bad" and "AG")

Materials:Cells grown in test tubes, Miniprep Kit, glycerin, centrifuge

Method:Glycerol stock: 500 µL of 100 % Glycerin + 500 µL of the liquid overnight cultureMiniPrep: according to the manual

Results:
ready plasmids AID(BBa_K103001) and pcdna5/frt per competent cell line ("good", "bad" and "AG")* location: Plasmids: AID(BBa_K103001)- 4th drawer in the -20 °C freezer, pcdna5/frt per competent cell line ("good", "bad" and "AG")-2nd drawer in the -20 °C freezerGlycerolstocks-box in -80 °C freezer marked with the green tape with a label(BM 28.6.2012, iGEM, AID, pcdna5/FRT)
Further tasks:
Gel electrophoresis for AID to check if it is intact.==Antikörper=====

2012-06-11

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Topic: Culture of CHO-Flp-In Cells

Investigators: Stefan, Tarek

Aim: Thawing of CHO-Flp-In Cells

Date/Time: 2012-06-11,11-20:00

Materials:
* Cryostock of CHO-Flp-In Cells* Complete Medium (Ham´s F12 + 10% FBS + 1% Pen/Strep + 2 mM L-Glutamin)* Zeocin* 75 cm² culture flask
Method:
Growth and Maintenance of Flp-In Cell Lines (Invtrogen):* incubation of thawed cells in 12 ml complete medium without Zeocin for 3h* change of medium (get rid of DMSO) and incubation overnight 37°C , 5% CO2
Results:
* 1x 75cm² culture flask with attached cells
Further tasks:
* change medium against medium with 100 µg/ml Zeocin (done on 2012-06-12)* change medium after 2-3 days (done on 2012-06-14)* get the cells to 80-90% confluence (daily check) for splitting===

2012-06-13

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Topic: Plasmid DNA Purification

Investigators: Kerstin, Maria

Aim: Purification of Plasmids pFRT/lacZeo,pcDNA5/FRT, pOG44

Date/Time: 2012-06-13,12:00-14.00

Materials: Macherey-Nagel Purification Kit
* E.coli XL-1 blue culture transformed with pFRT/lacZeo, pcDNA5/FRT, pOG44* 2 clones of each plasmid* 2 ml of each culture taken
Method: Plasmid purification (Miniprep)
Protocol-at-a-glance (Macherey-Nagel)* pellet of 2 ml from each culturevariation: centrifugation steps 1 min instead of 30 sec

Results:
Plasmids pFRT/lacZeo, pcDNA5/FRT, pOG44
* location: freezer -20°, Box2
Further tasks:
* measurement of DNA concentration* control with restriction digest===

2012-06-15

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Topic: Culture of CHO-Flp-In Cells

Investigators: Tarek

Aim: Splitting of CHO-Flp-In Cells

Date/Time: 2012-06-15, 14-16:00

Materials:
* 1 x 75 cm² flask with confluent CHO-Flp-In Cells* 8 x 75 cm² culture flasks* complete Medium with Zeocin* PBS* Trypsin/EDTA
Method:
Growth and Maintenance of Flp-In Cell Lines (Invitrogen):* remove medium and wash the cells with 10 ml PBS* Add 2 ml Trypsin/EDTA solution (ca. 3 min ´til the cells detached)* Add 8 ml of complete medium (+Zeocin), and briefly resuspend the cells* 1 x 2 ml of the cellsuspension in 13 ml complete medium (1:5 splitting)* 7 x 1 ml of the cellsuspension in 14 ml complete medium (1:10 splitting)* incubation 37°C, 5% CO2
Results:
* 1 x 1:5 CHO-Flp-In Cells* 7 x 1:10 CHO-Flp-In CellsFurther tasks:
* get the cells to 90% confluence* freezing cells===

2012-06-19

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Topic: Culture of CHO-Flp-In Cells

Investigators: Stefan, Tarek

Aim: Freezing and passaging cultured CHO-Flp-In Cells

Date/Time: 2012-06-19, 10-12:00

Materials:
* confluent CHO-Flp-In Cells* Complete Medium* Freezing Medium (90% Complete Medium + 10% DMSO)* PBS* Trypsin/EDTA* Neubauerzählkammer* Falcon-tubes (15ml + 50ml)
Method:
Growth and Maintenance of Flp-In Cell Lines (Invitrogen);Freezing Cells:* prepare 20 ml of Freezing medium; label cryovials* trypsinate the cells (like 2012-06-15); 5 x 75 cm² flasks* counting the cells in Neubauerzählkammer = 2,3 x 10e6/ml = 2,3 x 10e7/flask = 5 Cryostocks per flask á 4,6 x 10e6 cells (min 3 x 10e6 cells/ml)* centrifugate the cells in two 50 ml falcon tubes (á 25 ml) and aspirate off the medium* resuspend the cellpellets in 10 ml freezing medium* add 1 ml cell suspension into one cryovial (x20)* place the vials in a styroporbox and freeze them in -80°C Freezer* passaging cells (2 x 75 cm² flasks) was done like on 2012-06-15
Results:
* 19 cryovials with CHO-Flp-In Cells* 2 x 1:10 CHO Flp-In CellsFurther tasks:
* reactivate one cryostock===

2012-06-20

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Topic: Culture of CHO-Flp-In Cells

Investigators: Tarek

Aim: Reactivate/Thawing CHO-Flp-In Cryostock

Date/Time: 2012-06-20, 12-13

Materials:
* Cryostock of CHO-Flp-In Cells* Complete Medium (Ham´s F12 + 10% FBS + 1% Pen/Strep + 2 mM L-Glutamin)* Zeocin* 75 cm² culture flask
Method:
Growth and Maintenance of Flp-In Cell Lines (Invitrogen):
changes to 2012-06-11:* resuspend the thawed cells in 12 ml complete medium and centrifugate (to save 3h), 250 g, 5 min, RT* aspirate off the medium* resuspend the cells in complete medium (5ml)* place the resuspended cells in a culture flasks with 10ml complete medium + 15 µl Zeocin
Results:
* 1 x 75 cm² flasks with reactivated crystock of CHO-Flp-In CellsFurther tasks:
* change medium* get reactivated cryostock to confluence* splitting cells* freeze second charge of cells===

2012-06-22

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Topic: Culture of CHO-Flp-In Cells

Investigators: Tarek

Aim: Splitting Cells for 2nd freezing charge

Date/Time: 2012-06-22, 15-16:00

Materials:
* confluent CHO-Flp-In Cells (reactivated cryostock)* 5 x 75 cm² culture flasks* complete medium + Zeocin* PBS* Trypsin/EDTA
Method:
like 2012-06-15
Results:
* 5 x 75 cm² CHO-flp-In CellsFurther tasks:
* get cells 90% confluence* freeze cells===

2012-06-22

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Topic: Planning the antibody construct

Investigators: Maria, Sascha

Aim: Draft for gene construct, searching appropriate Fc-part

Date/Time: 2012-06-22, 14:00 - 16:30

Materials:
* Databases* Paper
Method:
reviewing of sequences

Results:
* nucleotide sequence of human C-kappa1 gene
Further tasks:
* further search for sequences===

2012-06-27

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Topic: Planning the antibody construct

Investigators: Sascha, Maria

Aim: Draft for gene construct

Date/Time: 2012-06-28,17:30-23:45

Materials:
* Databases* Geneious* Paper
Method:
Planning and reviewing of sequence

Results:
* antibody construct with exon/intron structure
Further tasks:
* meeting with Dr. Kappel (Monday) and Prof. Lenhard (Wednesday) for clarification of exon/intron structure* further control* ordering of gene synthesis

Topic: Primer-Design for GATC-sequencing of pcDNA5-FRT and pOG44; vectors for stable transfection

Investigators: Sascha, Maria, Tarek

Aim: Primer-Design for pcDNA5-FRT and pOG44

Materials:
* lablife: sequences of invitrogen vectors pcDNA5-FRT and pOG44* Oligocalc to calculate Tm of primers based on GATC-requirements* GeneiousResults:
* forward-primer for CMV-promotor in pcDNA5-FRT and pOG44* reverse-primer for pcDNA5-FRT in hygromycin at N-terminus* reverse-primer for pOG44 in N-terminus of flp-gene
Further tasks:
* ordering of primers at GATC* preparation of pcDNA5-FT, pOG44 and ordered primers based on requirements of GTAC==Virus=====

2012-06-07

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Topic: Primer design -Cloning: Sortase-Tag an VP2 + PCR for VP2 with Kozak-sortase_tag-myc_tag

Investigators: Kathi, Laura, Tobias, Xenia
Aim: Primer design of VP2 region
Materials:* Vector:P10_pSB1C3_001_CMV_DARPin_ML_VP2/3_587KO_6xHis* Sortase-sequence: amino acid sequence: LTAPG translated to gene sequence: CTTACAGCCCCAGGA* restriction sites: NgoMIV
Method: Geneious
Results:
* f_Primer_preNgoMIV+SortaseMotiv1+VP2* r_Primer_VP2_1
Further tasks:*check the primerChanges:
* primer for cmv region:* include restriction sites SpeI and XbalI, remove NgoMIV===

2012-06-14

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Topic: Primer design - Cloning: Sortase-Tag an VP2 + PCR for VP2 with Kozak-sortase_tag-myc_tag

Investigators: Kathi, Laura, Tobias, Xenia
Aim:Change primer of 2012-06-07, generate primers for the cmv region + cmv-suffix with SpeI restriction site, include XbalI restriction site in VP2-prefix include myc-tag
Materials:* Vector:P10_pSB1C3_001_CMV_DARPin_ML_VP2/3_587KO_6xHis* Sortase-sequence: amino acid sequence: LTAPG translated to gene sequence: CTTACAGCCCCAGGA* Kozak-sequence: gccgcc* restriction sites: SpeI and XbalI
Method: Geneious
Results:* f_Primer_preXba1 with overhang of: kozak+SortaseMotiv1+myc-tag+VP2 (forward primer)* r_Primer_VP2_1 (reverse primer)* f_Primer_XbaI+cmv (forward primer)* r_Primer_CMV+suf (reverse primer)* location: directory: VIRUS/complete
Further tasks:
* control* improvements* order===

2012-06-19

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Topic: Primer design - Cloning: Sortase-Tag linked on VP2 + PCR for VP2 with Kozak-sortase_tag-myc_tag

Investigators: Kathi, Laura, Tobias, Xenia
Aim: change primers of 2012-06-14
Materials:* vector:P10_pSB1C3_001_CMV_DARPin_ML_VP2/3_587KO_6xHis* sortase-sequence: amino acid sequence: LTAPG translated to gene sequence: CTTACAGCCCCAGGA* kozak-sequence: gccgcc* restriction sites: SpeI and XbalI
Method:Geneious
Results:* f_Primer_preXba1 with overhang: kozak+SortaseMotiv1+myc-tag+VP2 (forward primer)* r_Primer_VP2_1-Temp. 80.3 °C (reverse primer)* f_Primer_XbaI+Überhang_cmv (forward primer)* r_Primer_CMV+suf (reversed)+ overhang (reverse primer)* location: directory: VIRUS/complete
Further tasks:
* control* improvements===

2012-06-22

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Topic: Primer design - Cloning: Sortase-Tag an VP2 + PCR for VP2 with Kozak-sortase_tag-myc_tag

Investigators: Kathi, Laura, Tobias, Xenia
Aim:* change primer of 2012-06-19* trim primer: r_Pst_Primer_VP2_1+overhang-to reduce temperature uo to 68 °C* remove primers for cmv region
Materials:* Vector: p10_pSB1C3_001_CMV_DARPin_ML_VP2/3_587KO_6xHis* Sortase-sequence: amino acid sequence: LTAPG translated to gene sequence: CTTACAGCCCCAGGA* Kozak-sequence: gccgcc* restriction sites: XbaI in forward primer for VP2 region
Method:Geneious
Results:* prf_XbaI_kozak_SortaseMotivN_myc_VP2 (forward primer)* prr_VP2_PstI_Temp68 (reverse primer)* location: directory geneious VIRUS/complete
Further tasks:
* control of the primer===

2012-06-28

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Topic: TAE-buffer

Investigator:Xenia
Aim: TAE-buffer(50x)- 1l
Materials:
242 g tris base
57.1 mL glacial acetic acid
100 mL 0.5 M EDTA
add 1 L Millipore -water
Further tasks:*Agarose gel electrophoresis===

2012-06-29

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Topic: Primer solution

Investigator:Kathi
Aim: primer solution in 100 µM
Materials:* prr_VP2_PstI_Temp68 ad 394 µL Aqua dest* prf_XbaI_kozak_SortaseMotivN_myc_VP2 ad 178 µL Aqua dest
Results:* prf_XbaI_kozak_SortaseMotivN_myc_VP2 --> c=100 µM* prr_VP2_PstI_Temp68 --> c=100 µMFurther tasks:* PCR