Team:Potsdam Bioware/Lab/Labjournal/June

AID-Group

1st Labday 2012-06-09

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)

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:

• reverse primer without NES

Further tasks:

• design of the forward primer

• design of the practice part

2012-06-24

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.

2012-06-25

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 Freezer

Further tasks:

• testing ability for transformation of competent cells

2012-06-27

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

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 culture

MiniPrep: 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 freezer

Glycerolstocks-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.

Antibody

2012-06-11

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

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 culture

variation: 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

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 Cells

Further tasks:

• get the cells to 90% confluence

• freezing cells

2012-06-19

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 Cells

Further tasks:

• reactivate one cryostock

2012-06-20

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 Cells

Further tasks:

• change medium

• get reactivated cryostock to confluence

• splitting cells

• freeze second charge of cells

2012-06-22

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 Cells

Further tasks:

• get cells 90% confluence

• freeze cells

2012-06-22

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

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

• Geneious

Results:

• 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

[edit]

2012-06-07

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 primer

Changes:

• primer for cmv region:
• include restriction sites SpeI and XbalI, remove NgoMIV

[edit]

2012-06-14

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

[edit]

2012-06-19

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

[edit]

2012-06-22

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

[edit]

2012-06-28

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

[edit]

2012-06-29

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 µM

Further tasks:

• PCR