Team:Potsdam Bioware/Lab/Labjournal/August

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AID

2012-08-01

glycerol stocks & miniprep of eGFP and GFP

Investigators: Tom S.

Time:

2012-07-03 7:30 am

Materials:

Glycerol, Miniprep Kit, overnight cultures (eGFP; GFP)

Method:

Glycerol stock: 300 µL Glycerol 99,8 % + 700 µL over night cultures --> put in -80 °C freezer

Miniprep of overnight cultures (eGFP in pSB1C3 (BBa_K404316); 2 clones of GFP in pSB1A2 (BBa_E0040))

Results:

DNA concentrations via nanodrop:

eGFP = 335,6 ng/µL

GFP 1 = 291,5 ng/µL

GFP 2 = 261,7 ng/µL

Further tasks:

preparative digestion with eGFP and CMV+mod. AID (without NES, with NLS)

Topic: Ligation CMV & modified AID (without NES, with NLS) in pSB1C3 with hGH-PolyA

Investigators:

Chris

Aim:

Ligation of CMV & modified AID in pSB1C3(backbone) and hGH-polyA (insert)

Materials:

T4 DNA-Ligase, T4-Ligase-buffer,
samples:

CMV+PCR1C3C2(3318 nt, MW=2048.48 kDa) : 188.1 ng/µl (91.8 nM)

CMV+PCR2C2C1 : 182.2 ng/µl (88.9 nM)

hGH-polyA (495 nt, MW=305.4 kDa): 26.1 ng/µl (85 nM)

Method:

DNA fragment ligation: according to the manual

sample preparation 1:

2 µL CMV+PCR1C3C2 c=188.1 ng/µl (91.8 nM) ->

6 µL hGH-polyA c=26.1 ng/µl (85 nM) ->

1 µL T4 DNA-ligase

1 µL T4 DNA-ligase buffer

sample preparation 2:

2 µL CMV+PCR2C2C1 c=182.2 ng/µl (88.9 nM) ->

6 µL hGH-polyA c=26.1 ng/µl (85 nM) ->

1 µL T4 DNA-ligase

1 µL T4 DNA-ligase buffer

incubation of samples for 1,5 h at room temperature

Results:

ligated plasmids(final construct mod.-AID (CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA, without eGFP) for transformation

Further tasks:

Transformation

Topic: transformation of ligated samples (CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA, without eGFP)

Investigators: Chris

Time: 2012-08-01 3 pm

Materials:

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

Method:

Transformation via manual, 10 µl of ligation samples were used

Plate incubation start: 13:30 pm

Results:

ready mutants to pick clones

Further tasks:

picking clones

Topic: preparative digestion

Investigators: Chris

Time: 2012-08-01

Materials:

Plasmids: pSB1C3 with CMV+PCR1C3C2 and CMV+PCR2C2C1 and eGFP

Restriction enzymes (SpeI, NgoMIV and AgeI)

NE4-buffer

Method:

sample preparation: each DNA 25 µL + 3 µL NE4-buffer + 1 µL SpeI + 1 µL AgeI (for CMV+AID+Kozak sequence) or 1µL NgoMIV (for eGFP)

incubation of samples for 3,5 h at 37 °C

Further tasks:

gel electrophoresis

Topic: Separation of cut DNA fragments via gel electrophoresis

Investigators: Tom S.

Time: 2012-08-01

Aim: Separation of cut DNA fragments via gel electrophoresis

Materials:

gel electrophoresis material

cut samples:

CMV+PCR1C3C2: Restriction enzymes (AgeI, SpeI); NEB buffer 4

CMV+PCR2C2C1: Restriction enzymes (AgeI, SpeI); NEB buffer 4

eGFP: Restriction enzymes (NgoMIV, SpeI); NEB buffer 4

Method:

samples:

- 30 µL AID cut with XbaI and PstI + 7,5 µL loading dye

gelelectrophoresis conditions:

30 µL of each samples into one big well

V = 90 V

duration roughly 75 minutes

Results:

Marked fragments were cut out of the gel and transferred into 1,5 mL Eppendorf tubes

Further Tasks:

Gel extraction

Investigators:

Tom S.

Aim:

Gel extraction of CMV + mod. AID + backbone and eGFP-insert

Materials:

centrifuge, Nucleo Spin and PCR clean up - Kit, heat block, nanodrop

Method:

DNA extraction: according to the manual

Results:

DNA concentrations via nanodrop:

CMV+PCR1C3C2+backbone = 158,1 ng/µL -> 77,1 nM (with mass conc. of 2050,94 kDa)

CMV+PCR2C2C1+backbone = 232,0 ng/µL -> 113,1 nM (with mass conc. of 2050,94 kDa)

eGFP = 55,8 ng/µL -> 123,4 nM (with mass conc. of 452,3 kDa)

location: -20 °C freezer, topmost drawer

ready DNA for ligation

Further tasks:

ligation of fragments

2012-08-02

Ligation of CMV+AID without NES, with NLS+Kozak sequence+backbone and eGFP

Investigators:

Tom S.

Aim:

Ligation of CMV+AID without NES, with NLS+Kozak sequence+backbone and eGFP

Materials:

T4 DNA-Ligase, samples(CMV+PCR1C3C2+backbone, CMV+PCR2C2C1+backbone,eGFP)

Method:

DNA fragment ligation: according to the manual

1st sample preparation:

4 µL (eGFP) c=55,8 ng/µL(123,4 nM)-> 49,4 nM

2 µL (CMV+PCR1C3C2+backbone) c=158,1 ng/µL(77,1 nM) -> 15,4 nM

1 µL (T4 DNA-Ligase)

1 µL 10x T4 DNA Ligase Buffer

2 µL (DNase free water)

2nd sample preparation:

4 µL (eGFP) c=55,8 ng/µL(123,4 nM)-> 49,4 nM

2 µL (CMV+PCR2C2C1+backbone) c=232,0 ng/µL(113,1 nM) -> 22,6 nM

1 µL (T4 DNA-Ligase)

1 µL 10x T4 DNA Ligase Buffer

2 µL (DNase free water)

incubation of sample for 1,5 h at 22°C

Results:

ready DNA for transformation

location: -20 °C freezer, topmost drawer

Further tasks:

Transformation

Topic: Transformation of ligated samples - CMV+AID without NES, with NLS+Kozak sequence+eGFP

Investigators: Tom S. , Chris

Time: 2012-08-02 3 pm

Materials:

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

ligated samples (CMV+PCR1C3C2+eGFP in pSB1C3, same consruct with PCR2C2C1)

Method:

Transformation via manual, 10 µl of ligation samples were used

Plate incubation start: 14:00 pm

Results:

ready for growing mutants to pick clones

Further tasks:

picking clones

Topic: picking clones & inoculation in 5 mL LB of CMV+AID without NES, with NLS+hGH-PolyA in pSB1C3

Investigators: Chris

Method:
picking clones (1 per plate -->4) CMV + modified AID(PCR1C3C2&PCR2C2C1) +hGH(PolyA)in pSB1C3 and inoculation in 5 ml LB medium + 5µl chloramphenicol stock, shaking over night at 37°C, 300 rpm, approx. 16 hours

Further tasks:

glycerol stocks & Miniprep

2012-08-03

glycerol stocks & miniprep of CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA, without eGFP plasmids

Investigators: Chris

Time:

2012-08-03 8:00 am

Materials:

Glycerol, Miniprep Kit, (CMV+PCR1C3C2/PCR2C2C1+polyA)

Method:

Glycerol stock: 300 µL Glycerol 99,8 % + 700 µL over night cultures --> put in -80 °C freezer

Mini Prep over night cultures (CMV+PCR1C3C2-Nr1+polyA in pSB1C3,same construct with PCR1C3C2-Nr2, PCR2C2C1-Nr1,PCR2C2C1-Nr2)

Results:

transfection ready biobricks: CMW+ modified AID+hGH-polyA

DNA - concentrations via nanodrop:

CMV+PCR1C3C2-Nr1+polyA in pSB1C3= 628 ng/µL

CMV+PCR1C3C2-Nr2+polyA in pSB1C3= 577.5 ng/µL

CMV+PCR2C2C1-Nr1+polyA in pSB1C3= 590.8 ng/µL

CMV+PCR2C2C1-Nr2+polyA in pSB1C3= 571.4 ng/µL

Further tasks:

sequencing

Overnight culture of CMV+AID without NES, with NLS+Kozak sequence+eGFP

Investigators: Chris, Tom S.

Time: 2012-08-03 5 pm

Materials:

LB medium, chloramphenicol 25 mg/ ml stock solution, plates with E. coli XL1 blue with plasmids: CMV+PCR1C3C2C1-2+eGFP and CMV+PCR2C2C1C1-2+eGFP

Method: picking clones(1 per plate) and inoculation in 5 ml LB medium + 5µl chloramphenicol stock shaking over night at 37°C, 300 rpm, approx. 16 hours

Further tasks:

glycerol stocks & Miniprep

2012-08-04

glycerol stocks & miniprep

Investigators: Basia

Time:

2012-08-04 10:00 am

Materials:

Glycerol, Miniprep Kit, overnight culture

Method:

Glycerol stock: 300 µL Glycerol 99,8 % + 700 µL overnight cultures --> put in -80 °C freezer

Miniprep overnight cultures (CMV+PCR1C3C2-Nr1+eGFP in pSB1C3,same construct with PCR1C3C2-Nr2, PCR2C2C1-Nr1,PCR2C2C1-Nr2)

Results:

transfection ready biobricks: CMV+ AID without NES, with NLS+Kozak sequence+ eGFP

DNA - concentrations via nanodrop:

CMV+ AID+eGFP 1 in pSB1C3= 386,8 ng/µL

CMV+ AID+eGFP 2 in pSB1C3= 404,9 ng/µL

CMV+ AID+eGFP 3 in pSB1C3= 442,6 ng/µL

CMV+ AID+eGFP 4 in pSB1C3= 377,4 ng/µL

Further tasks:

digestion

2012-08-06

Topic: preparative digestion

Investigators:Tom S.

Time: 2012-08-06

Materials:

pSB1C3 Vector with CMV+AID without NES, with NLS+Kozak sequence+eGFP (SpeI, PstI; Fast Digest); Fast Digest Green Buffer

Method:

preparative digestion: 25 µL DNA + 1 µL of each enzyme + 3 µL Fast Digest Green Buffer (incubation for 2,5 h)

Gel electrophoresis & Gel extraction

Investigators: Rico, Tom S., Chris

Gel extraction:
final concentrations:
CMV+PCR???1+eGFP-cutS+P(4035 nt, MW=2492 kDa) : 246 ng/µl (98.7 nM)

CMV+PCR???2+eGFP-cutSCMV-cutS+P: 255 ng/µl (102.3 nM)

CMV+PCR???3+eGFP-cutSCMV-cutS+P: 187.6 ng/µl (75.3 nM)

CMV+PCR???4+eGFP-cutSCMV-cutS+P: 178.6 ng/µl (71.7 nM)

Further Tasks: Ligation, Transformation

2012-08-07

Ligation

Investigators: Chris

Aim:

Ligation of CMV + AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3(backbone) and hGH-polyA (insert)

Materials:

T4 DNA-Ligase, T4-Ligase-buffer,
samples:

CMV+PCR???1+eGFP-cut S+P(4035 nt, MW=2492 kDa) : 246 ng/µl (98.7 nM

CMV+PCR???2+eGFP-cut S+P (4035 nt, MW=2492 kDa) : 182.2 ng/µl (88.9 nM)

CMV+PCR???3+eGFP-cut S+P: 187.6 ng/µl (75.3 nM)

CMV+PCR???4+eGFP-cut S+P: 178.6 ng/µl (71.7 nM)

hGH-polyA (495 nt, MW=305.4 kDa): 26.1 ng/µl (85 nM)

hGH-polyA (cut:Xba1 and Pst1) c=48.5 nM

Method:

DNA Fragment ligation: according to the manual

sample preparation 1-4:

1 µL CMV+PCR1C3C2 c=188.1 ng/µl (91.8 nM) ->

3 µL hGH(polyA) c=26.1 ng/µl (85 nM) ->

1 µL T4 DNA-ligase

1 µL T4 DNA-ligase buffer

4 µL H₂0

incubation of samples for 1,5 h at room temperature

Results:

ligated plasmids(CMV + AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA) for transformation

Further tasks:

Transformation

alignment of the complete WT AID and Sequencing

Investigators: Rico, Tom S.

Aim:

alignment of the complete WT AID and Sequencing (checking the quality of the clones)

Materials:

6 forward sequences of WT AID from GATC and 6 reverse sequences of the same fragment,

draft sequences, Geneious

Results:

1., 3., 5. and 6. clone: mutation of the 3rd nucleotide in front of the AID startcodon (is good);

2. and 4. clone: frameshift (loss of the 155th nucleotide (2nd clone) or loss of the 239th nucleotide (4th clone))

Further tasks:

discard the mutated clones and preparation for transfection of the final BioBrick BBa_K929000

alignment of the mod. AID PCR-product and Sequencing

Investigators: Rico, Tom S.

Aim:

alignment of the mod. AID PCR-product and Sequencing (checking the quality of the clones)

Material:

6 forward sequences of mod. AID PCR-product from GATC and 6 reverse sequences of the same fragment,draft sequences, Geneious

Results:

all vectors have no PCR-insert in the backbone (instead CMV)

Further tasks:

discard all samples and clones, made a eGFP and PCR-insert fusion with XbaI, NgeMIV and AgeI digestion, design new primer with PstI recognition site

2012-08-08

Planing cooperation -> Fusion of AID without NES with TAL-Protein, primer design

Investigators: Chris, Rico,

AID will be fused to the TAL-protein (N--TAL-AID--C) with BpiI.We will get the fusion protein in an eukaryotic expression-vektor (with CMV promoter or hopefully a weaker one, depending on Team-Freiburg).

The 14 bp sequence for the TAL-protein has to have the following properties: (done by Antibody team)

5' positon 0- must be T,
positon 1- no T ,
Position 2- no A,
Position 13- no G, position 14- must be G

There are two of these sequences in the VH-region of the sc-FV-anti EGFR 425 but not in the direct neighborhood of CDR3.

Planing BBa_K929001 and BBa_K929003

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

Aim: planing how to digest and ligate the vectors for BBa_K929001 and BBa_K929003

Material: Geneious

Results 1:

pSB1C3 with eGFP -> cut with NgoMIV and XbaI, 2793 bp - pSB1C3 backbone+eGFP, 12 bp - rest

PCR-amplificate (without PstI restriction site) -> cut with AgeI and XbaI, 589 bp - modified AID insert (AID without NES, with NLS+Kozak sequence)

Results 2:

pSB1C3 with CMV -> cut with PstI and XbaI, 2061 bp - pSB1C3 backbone, 680 bp - rest

PCR-amplificate (with PstI restriction site) -> cut with PstI and XbaI, 613 bp - modified AID insert (AID without NES, with NLS+Kozak sequence)

Further tasks:

design and ordering of primers, practical part

500px

Primer design and ordering for BBa_K929001

Investigators: Tom S., Rico

Time: 2012-08-08

Primer (reverse, complement)with AgeI, SpeI and PstI recognition site:

GCCTGCAGCGGCCGCTACTAGTATTAACCGGTGGGCAAAAGGATGCGCCGAAGC

Primer design and ordering for sequencing BBa_K929003

Investigators: Tom S., Rico

Time: 2012-08-08

Primer bind on mod. AID Sequence:

TTTCAAAGCCTGGGAAGG

Primer (reverse, complement)bind on eGFP sequence:

GTGCCCATTAACATCACC

PCR of AID without NES, with NLS+ Kozak sequence

Investigators:

Rico

Aim:

amplification of the AID and insertion of Kozak sequence and NLS sequence

Materials:

Phusion, template (AID insert), Primers designed by Tom S. and Rico on 12.07.2012 , dNTPs, Polymerase)

PCR clean-up kit

Method:

polymerase chain reaction

Mastermix

reagent	volume [µL]
HF Phusion buffer 5x	10
dNTPs	1
Primer (Forward)	1,25
Primer (Reverse)	1,25
DNA (Plasmid)	1,0
Phusion Polymerase	0,5
water	35,0

Program

step	Temperature [°C]	duration [s]	cycles
denaturation	98	30	1
denaturation	98	5	17
annealing + elongation	72	45	17
denaturation	98	5	17
elongation	72	25	17
final elongation	72	600	1
cooling	4	∞	1

Results:
112 ng/µl - 1st sample, 113,8ng/µl 2nd sample, 22,1 negative control

Further tasks:

digestion + agarose gel electrophoresis

Digestion of amplified AID without NES, with NLS+Kozak sequence and GFP and separation with gel electrophoresis

Investigators:Rico

Aim: digestion of mod.-AID with XbaI and AgeI and GFP with NgoMIV and XbaI and separation via gel electrophoresis

Results:

Gel extraction of eGFP + pSB1C3 backbone and AID without NES, with NLS+Kozak sequence insert

Investigators:

Tom S.

Aim:

Gel extraction of eGFP + pSB1C3 backbone and AID without NES, with NLS+Kozak sequence insert

Materials:

centrifuge, Nucleo Spin and PCR clean up - Kit, heat block, nanodrop

Method:

extract DNA: according to the manual

Results:

DNA-concentrations via nanodrop:

eGFP + pSB1C3 backbone = 59,1 ng/µL -> 34 nM

AID without NES, with NLS+Kozak sequence 1 = 22,8 ng/µL -> 62 nM

AID without NES, with NLS+Kozak sequence 2 = 25,2 ng/µL -> 69.3 nM

location: -20 °C freezer, topmost drawer

ready DNA for ligation

Further tasks:

ligation of fragments

2012-08-09

Ligation of insert: PCR-Product (AID without NES, with NLS+Kozak sequence cut: XbaI, AgeI) & backbone: eGFP in pSB1C3 (cut: NgoMIV, Xba1)

Investigators:

Chris

Aim:

Ligation of insert: PCR-Product (AID without NES, with NLS+Kozak sequence cut: XbaI, AgeI) & backbone: eGFP in pSB1C3 (cut: NgoMIV, Xba1)

Materials:

T4 DNA-Ligase, T4-Ligase-buffer,
samples:

eGFP + pSB1C3 backbone(2793bp, MW=1725.2 kDA) = 59,1 ng/µL -> 34 nM

PCR-product 1 (598 bp, MW=363.4 kDA)= 22,8 ng/µL -> 62 nM

PCR-product 2 (598 bp, MW=363.4 kDA)= 25,2 ng/µL -> 69.3 nM

Method:

DNA Fragment ligation: according to the manual

sample preparation (same for both samples):

1 µL eGFP + pSB1C3 backbone 59,1 ng/µL -> 34 nM

2 µL PCR-product 22,8 ng/µL -> 62 nM

5 µl wather

1 µL T4 DNA-ligase

1 µL T4 DNA-ligase buffer

incubation of samples for 1,5 h at room temperature

Results:

ligated plasmids(final construct AID without NES, with NLS+Kozak sequence+eGFP) for transformation

Further tasks:

Transformation

Topic: transformation of ligated samples (AID without NES, with NLS+Kozak sequence+eGFP)

Investigators: Chris

Time: 2012-08-09 5 pm

Materials:

samples: ligated AID without NES, with NLS+Kozak sequence 1+eGFP in pSB1C3, ligated AID without NES, with NLS+Kozak sequence 2+eGFP in pSB1C3

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

Method:

Transformation via manual, 10 µl of ligated samples were used

Plate incubation start: 5:00 pm

Results:

ready mutants to pick clones

Further tasks:

picking clones

2012-08-10

PCR of AID to create AID without NES, with NLS+Kozak sequence

Investigators:

Tom S.

Time: 2012-08-10 8 am

Aim:

PCR of AID to create AID without NES, with NLS+Kozak sequence

Method:

Mastermix

reagent	volume [µL]
HF Phusion buffer 5x	10
dNTPs	1
Primer (Forward)	1,25
Primer (Reverse)	1,25
DNA (Plasmid)	1,0
Phusion Polymerase	0,5
water	35,0

Program

step	Temperature [°C]	duration [s]	cycles
denaturation	98	30	1
denaturation	98	5	17
annealing + elongation	72	45	17
denaturation	98	5	17
elongation	72	25	17
final elongation	72	600	1
cooling	4	∞	1

PCR-clean up, final concentrations: spektra inconsistent, A260/280 very low(maybe contamination, problem with clean up):

negativ control 1: 97.4 ng/µL (spektrum inconsistent)

negativ control 2: 108.3 ng/µL (spektrum inconsistent)

PCR product 1-1: 73.1 ng/µL (spektrum inconsistent but somehow looked like a DNA curve)

PCR product 1-2: 141.5 ng/µL (spektrum inconsistent)

PCR product 2-1: 222 ng/µL (spektrum inconsistent)

PCR product 2-2: 162.2 ng/µL (spektrum inconsistent)

test digestion of PCR product(AID without NES, with NLS+Kozak sequence) with PmlI

Investigators: Rico, Chris

Materials:

samples: negative control and PCR1-1 (AID without NES, with NLS+Kozak sequence)

FastDigest PmlI

10x FD Green Buffer

sterile water

Method:

30 µl mix: 1 µL negative control, 1µL PmlI, 3µL 10x FD Green Buffer, 25 µL sterile water

30 µl mix: 2 µL negative control, 1µL PmlI, 3µL 10x FD Green Buffer, 24 µL sterile water

30 µl mix: 2 µL PCR product 1, 1µL PmlI, 3µL 10x FD Green Buffer, 24 µL sterile water

30 µl mix: 3 µL PCR product 1, 1µL PmlI, 3µL 10x FD Green Buffer, 23 µL sterile water

digestion incubated at 37°C for 2 h 30 min

further tasks:

gel electrophoresis

Gel electrophoresis to control the test digestion of the PCR-product (AID without NES, with NLS+Kozak sequence)

Investigators: Rico

Time: 2012-08-11;

Materials:

gel electrophoresis equipment

digested samples

Method:

loading slots with 30 µL digested sample (digested samples and 7,5 µL Loading dye)

standard gel electrophoresis procedure

Results:

(sizes were calculated with ImageJ)

Further tasks:

sequencing

planing the AID phage display project

Investigators: Tom S.,Rico , Chris

Inoculation of AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3, pBAD-mYFP Venus (phage display: arabinose promoter-vector), CREB-PYP UT156- sSD-pAK100 (phage display: vector for antibody-P3-fusion)

Investigators: Chris

Time: 2012-08-10

Materials:

LB medium

Chloramphenicol 25 mg/ ml stock solution

Plasmids: pSB1C3 with AID without NES, with NLS+Kozak sequence(PCR1&2)+eGFP-chloramphenicol resistance, pBAD-mYFP Venus-ampicillin resistance, CREB-PYP UT156- sSD-pAK100-chloramphenicol resistance

Method:

Inoculation of:

1 culture pSB1C3 with AID without NES, with NLS+Kozak sequence(PCR1)+eGFP from plate in 5 ml LB medium + 5µL chloramphenicol

1 culture pSB1C3 with AID without NES, with NLS+Kozak sequence(PCR2)+eGFP from plate in 5 ml LB medium + 5µL chloramphenicol

2 cultures pBAD-mYFP Venus from cryostock in 5 ml LB medium + 5µL ampicillin

2 CREB-PYP UT156- sSD-pAK100 from cryostock in 5 ml LB medium + 5µL chloramphenicol

shaking over night at 37°C, 300 rpm, approx. 16 hours

Further tasks:

Miniprep

2012-08-11

glycerolstocks & miniprep of AID without NES, with NLS+Kozak sequence+eGFP

Investigators: Basia

Time:

2012-08-11 10:30 am

Materials:

Glycerol, Miniprep Kit, overnight culture

Method:

Glycerol stock: 300 µL Glycerol 80 % + 700 µL overnight cultures --> put in -80 °C freezer

Miniprep of overnight cultures (pAK 100, PCR1+eGFP im PSB1C3, PCR2+eGFP im PSB1C3, Arabinose promoter in pBad)

Results:

DNA concentrations via nanodrop:

pak 100 = 187,7ng/µl

PCR1+eGFP im PSB1C3 = 12,5ng/µl

PCR2+eGFP im PSB1C3 = 227,2 ng/µl

Arabinose promoter im pbad = 341,2 ng/µl

Further tasks:

test digestion

2012-08-13

PCR of AID to create AID without NES, with NLS+Kozak sequence with PstI

Investigators:

Tom S.

Time: 2012-08-13 11 am

Aim:

PCR of AID to create AID without NES, with NLS+Kozak sequence with PstI (in the next step the PCR-product will be digested to find out whether the PCR was successful or not)

Method:

Mastermix

reagent	volume [µL]
HF Phusion buffer 5x	10
dNTPs	1
Primer (Forward)	1,25
Primer (Reverse)	1,25
DNA (Plasmid)	1,0
Phusion Polymerase	0,5
water	35,0

Program

step	Temperature [°C]	duration [s]	cycles
denaturation	98	30	1
denaturation	98	5	17
annealing + elongation	72	45	17
denaturation	98	5	17
elongation	72	25	17
final elongation	72	600	1
cooling	4	∞	1

PCR-clean up, final concentrations:

PCR 1: 20,9 ng/µL

PCR 2: 145,1 ng/µL

PCR 3: 71,6 ng/µL

PCR 4: 32,4 ng/µL

preparative digestion, test digestion and gel electrophoresis

Investigators:Rico, Tom S., Chris

Materials:

samples: AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3, AID in pSB1C3, pBAD-mYFP Venus, CMV in pSB1C3, AID without NES, with NLS+Kozak sequence

FastDigest PmlI, XbaI, PstI, SpeI

10x FD Green Buffer

sterile water

Method:

30 µl mix: 2µL test digestion sample, 1µL PmlI, 3µL 10x FD Green Buffer, 24 µL sterile water

30 µl mix: 25 µL sample, 1µL each Enzyme, 3µL 10x FD Green Buffer

sample	Enzyme 1	Enzyme 2
pBAD-YFP Venus	XbaI	PstI
AID	XbaI	PstI
AID+eGFP	PmlI	-
AID-PCR-product AID without NES, with NLS+Kozak sequence	PmlI	-
AID-PCR-product	XbaI	PstI
CMV	XbaI	PstI
AID+eGFP	XbaI	PstI
CMV	SpeI	PstI

digestion incubated at 37°C for 2,5-5 h

results:

further tasks:

gel extraction

gel extraction

Investigators:Chris, Tom S.

Materials:

centrifuge, Nucleo Spin and PCR clean up Kit, heat block, nanodrop

Method:

extract DNA: according to the manual

Results:

sample	mass concentration	molecular weight	molar concentration
pBAD cut X+P	50.4 ng/µL	2484.7 kDA	20.28 nM
CMV cut: S+P	18.9 ng/µL	1681.32 kDA	11.24 nM
mod. AID1(with P) cut: X+P	65.4 ng/µL	378.3 kDA	172.9 nM
mod. AID4(with P) cut: X+P	14.8 ng/µL	378.3 kDA	39.1 nM
wt AID cut: X+P	7.0 ng/µL	386.28 kDA	18 nM
CMV cut: X+P	52.1 ng/µL	1272.52 kDA	40.9 nM

the samples could be confounded? we`ll have to control it via gel electrophoresis

Further tasks:

ligation of fragments

inoculation of 4 further cultures of AID without NES, with NLS+Kozak sequence+ eGFP in pSB1C3

Investigators: Chris

Time: 2012-08-13 5 pm

Materials:

LB medium

Chloramphenicol 25 mg/ ml stock solution

plates with cultures: AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 (from 2012.08.09)

Method:

Inoculation of:

2 cultures pSB1C3 with AID without NES, with NLS+Kozak sequence(PCR1)+eGFP per plate in 5 ml LB medium + 5µL chloramphenicol

(--> 4 cultures)

Further tasks:

Miniprep

2012-08-14

Plasmid isolation of AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3

Investigators:

Rico

Time: 2012-08-14 8 am

Aim:

Isolation of AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3

Method:

Miniprep: according to the manual

Results:

AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 1: 183.3 ng/µL

AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 3: 257.5 ng/µL

AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 4: 180.6 ng/µL

Further Tasks:

Digestion with PstI, XbaI (preparative digestion) and PmlI (test digestion)

Gel electrophoresis

Preparative digestion of isolated AID without NES, with NLS+Kozak sequence+eGFP with XbaI and PstI and test digestion of AID without NES, with NLS+Kozak sequence+eGFP with PmlI and gel electrophoresis

Investigators:

Chris, Rico

Time: 2012-08-14 8 am

Aim:

preparative and test digestion of AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3

Method:

Digestion with XbaI, PstI and PmlI, gel electrophoresis

Results:

Gel extraction of digested AID without NES, with NLS+Kozak sequence+eGFP

Investigators:

Rico

Method:

Gel extraction kit

Results:

AID without NES, with NLS+Kozak sequence+eGFP (digested with X and P): 135.2 ng/µL

Further Tasks:

Ligation of AID without NES, with NLS+Kozak sequence+eGFP with CMV-Promotor

Ligation of CMV and AID without NES, with NLS+Kozak sequence(+P)+eGFP1&4, pSB1C3 + AID without NES, with NLS+Kozak sequence(+P)+eGFP1&4 ,pBAD +wtAID

Investigators:

Chris

Materials:

T4 DNA-Ligase, T4-Ligase buffer, water, samples

Method:

DNA Fragment ligation: according to the manual

sample preparation:

Fragment 1(BB)	Fragment 2(insert)	T4 DNA-Ligase	T4 DNA-Ligase buffer	water
pBAD cut X+P (20.28 nM)	wt AID cut: X+P (18 nM)
2 µL	6 µL	1 µL	1 µL	0 µL
CMV cut: S+P (11.24 nM)	mod. AID1(with P)+eGFP cut: X+P (172.9 nM)
5 µL	1 µL	1 µL	1 µL	2 µL
CMV cut: S+P (11.24 nM)	mod. AID4(with P)+eGFP cut: X+P (39.1 nM)
4 µL	4 µL	1 µL	1 µL	0 µL
pSB1C3 cut: X+P (40.9 nM)	mod. AID1(with P)+eGFP cut: X+P (172.9 nM)
3 µL	2 µL	1 µL	1 µL	3 µL
pSB1C3 cut: X+P (40.9 nM)	mod. AID4(with P)+eGFP cut: X+P (39.1 nM)
2 µL	6 µL	1 µL	1 µL	0 µL

incubation of samples for 1,5 h at room temperature

Results:

ligated plasmids ready for transformation

Further tasks:

Transformation

Topic: Transformation of ligated samples AID without NES, with NLS+Kozak sequence(+P)+eGFP+CMV and pBad + wtAID

Investigators: Chris

Materials:

samples (see above): ligated pBAD +wtAID (ampR), BB+CMV+modAID(+P) from PCR 1&4 (chlR), pSB1C+ modAID(+P) from PCR 1&4 (chlR)

-> 2 plates per ligated sample +2 extra plates pBAD +wtAID (condensed water from the lid dripped on the plated cultures in the first two plates), all in all 12 plates

Bunsen Burner, Agar Plates with chloramphenicol & ampicillin (for pBAD +wtAID)

icebox

competent E. coli cells (XL 1 Blue)

Method:

Transformation via manual, 10 µl of ligated samples were used

Plate incubation start: 4 pm

Results:

ready mutants to pick clones

Further tasks:

picking clones

2012-08-15

Ligation and transformation of CMV (Insert + backbone) and AID without NES, with NLS+Kozak sequence+eGFP (insert)

Investigators:

Rico, Tom S.

Aim:

Ligation of CMV (Insert + backbone) and AID without NES, with NLS+Kozak sequence+eGFP (insert), later transformation in XL-1

Materials:

T4 DNA-Ligase, samples(CMV and AID)

Bunsen Burner, Agar Plate with Chloramphenicol, 37 °C heater, centrifuge, spoon for weight

ligated sample (compare last step 14.08-2012)

icebox

competent E. coli cells (XL 1)

Method:

DNA Fragment ligation: according to the manual

sample preparation:

5 µL (CMV Fragment)(MW: 1681,3 kDa) c=18,9 ng/µL(11,2 nM) -> 5,6 nM

1 µL (mod. AID+eGFP Fragment) (MW: 821,92 kDa) c=135,2 ng/µL(164,5 nM) -> 16,5 nM

1 µL (T4 DNA-Ligase)

1 µL (T4 DNA-Ligase Buffer)

2 µL (DNase free water)

Transformation via manual

Plate incubation start: 5 pm

incubation of sample for 1,5 h at 22 °C

Results:

location: -20 °C freezer, topmost drawer

Further tasks:

picking clones

inoculation of AID without NES, with NLS+Kozak sequence in pSB1C3, AID without NES, with NLS+Kozak sequence+CMV and pBAD+WT AID

Investigators: Tom S.

Time: 2012-08-15 5 pm

Materials:

LB medium

Chloramphenicol 25 mg/ ml stock solution

Ampicillin 100 mg/ ml stock solution

plates with cultures: AID without NES, with NLS+Kozak sequence+CMV in pSB1C3 (from 2012-08-14), AID without NES, with NLS+Kozak sequence in pSB1C3 and pBAD+WT AID

Method:

Inoculation of:

2 clones of every plate

Further tasks:

Miniprep and cryo stocks

Topic: Transformation of pKMEF425bla

Investigators: Chris

Materials:

1 µL pKMEF425bla Plasmid

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

Method:

Transformation via manual, 1 µl of pKMEF425bla Plasmid was used

Plate incubation start: 3:30 pm

Results:

2 plates (Chloramphenicol) with XL1 blue E. coli colonies carrying pKMEF425bla

Further tasks:

picking clones & inoculation (for our group and the antibody group)

PCR of AID to create AID with BpiI-restriction sides for TAL-AID fusion, cooperation with team Freiburg

Investigators:

Chris, Rico

Aim:

PCR of AID to create AID with BpiI-restriction sides for TAL-AID fusion, cooperation with team Freiburg

Method:

Mastermix

reagent	volume [µL]
HF Phusion buffer 5x	10
dNTPs	1
Primer (Forward)	1,25
Primer (Reverse)	1,25
DNA (BBa_K103001 AID in pSB1A2 10 ng/µl)	1,0
Phusion Polymerase	0,5
water	35,0

Program

step	Temperature [°C]	duration [s]	cycles
denaturation	98	30	1
denaturation	98	5	17
annealing	50	20	17
elongation	72	18	17
denaturation	98	5	17
annealing+elongation	72	18	17
final elongation	72	600	1
cooling	4	∞	1

PCR-clean up, final concentrations:

PCR TAL-AID1: 80.7 ng/µL, inconsistent curve- no DNA (also see gels below), can be discarded

PCR TAL-AID2: 202.6 ng/µL

PCR TAL-AID3: 123.3 ng/µL

PCR TAL-AID4: 124 ng/µL, inconsistent curve- no DNA (also see gels below), can be discarded

PCR TAL-AID ist just 5` XbaI BpiI AID (without NES) BpiI(reversed) PstI 3`(but will be fused with TAL-therefore this PCR-product is called PCR TAL-AID)

Position: -20°C freezer-extrabox "phagedisplay, cooperation"

test digestion of PCR TAL-AID 1-4 with PmlI and uncut PCR TAL-AID 1-4, Gelelectrophoresis

Investigators:

Chris, Tom S., Rico

Aim:

check whether the PCR worked

Method:

Gel electrophoresis of PCR TAL-AID 1-4 uncut & PCR TAL-AID 1-4 cut with PmlI

Results:

the lowest lane could be primer dimers, therefore, PCR TAL-AID 2 & 3 will be used for further cloning. 1 and 4 can be discarded. The Location is the box phagedisplay and cooperation (-20 °C freezer).

2012-08-16

Miniprep of overnight cultures of AID without NES, with NLS+Kozak sequence in pSB1C3, pBAD-wt AID and CMV-AID without NES, with NLS+Kozak sequence

Investigators:

Rico, Tom S.

Aim:

Miniprep of AID without NES, with NLS+Kozak sequence in pSB1C3, pBAD-wt AID and CMV-AID without NES, with NLS+Kozak sequence

Method:

Miniprep Thermo Scientific according to the manual

Results:

Concentrations

Sample	Concentration [ng/µL]
AID without NES, with NLS+Kozak sequence in pSB1C3 4-1-1	224.3
AID without NES, with NLS+Kozak sequence in pSB1C3 1-2-2	297.0
AID without NES, with NLS+Kozak sequence in pSB1C3 4-1-2	210.4
AID without NES, with NLS+Kozak sequence in pSB1C3 1-2-1	246.7
AID without NES, with NLS+Kozak sequence in pSB1C3 4-2-2	271.4
AID without NES, with NLS+Kozak sequence in pSB1C3 4-2-1	229.1
AID without NES, with NLS+Kozak sequence in pSB1C3 1-1-2	193.4
AID without NES, with NLS+Kozak sequence in pSB1C3 1-1-1	233.5
pBAD wt AID 2-1	143.7
pBAD wt AID 3-1	140.9
pBAD wt AID 4-2	211.6
pBAD wt AID 1-1	164.9
pBAD wt AID 3-2	156.6
pBAD wt AID 4-1	145.3
pBAD wt AID 2-2	114.6
pBAD wt AID 1-2	114.6
CMV + AID without NES, with NLS+Kozak sequence 4-1-2	254.3
CMV + AID without NES, with NLS+Kozak sequence 1-1-2	334.8
CMV + AID without NES, with NLS+Kozak sequence 1-2-1	228.7
CMV + AID without NES, with NLS+Kozak sequence 1-2-2	311.9
CMV + AID without NES, with NLS+Kozak sequence 1-1-1	352.9
CMV + AID without NES, with NLS+Kozak sequence 4-2-2	241.2
CMV + AID without NES, with NLS+Kozak sequence 4-1-1	346.5
CMV + AID without NES, with NLS+Kozak sequence 4-2-1	318.2

Further Tasks:

Test digestion of AID without NES, with NLS+Kozak sequence in pSB1C3 with PmlI, XpaI and PstI, and test digestion of CMV-AID without NES, with NLS+Kozak sequence with XbaI and PstI

test digestion of CMV+AID without NES, with NLS+Kozak sequence with XbaI and PstI and test digestion of pBAD+WT AID and AID without NES, with NLS+Kozak sequence in pSB1C3 with XbaI, PstI and PmlI, Gel electrophoresis

Investigators:

Tom S.

Aim:

check which clone is the right one

Method:

Gel electrophoresis of all cut samples

Results:

We chose 1-1-2, 4-2-1 and 4-2-2 of AID without NES, with NLS+Kozak sequence in pSB1C3; all pBAD+WT AID and nothing of CMV + AID without NES, with NLS+Kozak sequence for further preparation.

Further Tasks:

sequencing and further cloning of the complete constructs

inoculation of CMV+AID without NES, with NLS+Kozak sequence+eGFP

Investigators: Tom S.

Time: 2012-08-16 5 pm

Materials:

LB medium

Chloramphenicol 25 mg/ ml stock solution

plates with cultures: CMV+AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 (from 2012.08.15.)

Method:

Inoculation of:

2 clones of every plate

(--> 4 cultures)

Further tasks:

Miniprep and cryo stocks

2012-08-17

Miniprep of overnight cultures of CMV+AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3

Investigators:

Tom S.

Aim:

Miniprep of overnight cultures of CMV+AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3

Method:

Miniprep Thermo Scientific: according to the manual

Results:

1-1 = 391,3 ng/µL

1-2 = 372,1 ng/µL

2-1 = 409,7 ng/µL

2-2 = 422,7 ng/µL

Further Tasks:

Test digestion of these samples PmlI, XpaI and PstI

test digestion of CMV+AID without NES, with NLS+Kozak sequence+eGFP with XbaI, PmlI and PstI, Gel electrophoresis

Investigators:

Tom S.

Aim:

check which clone is the right one

Method:

digestion for 4h

Gel electrophoresis of all cut samples

Results:

We chose 2-1 and 2-2 of CMV+AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 for further preparation.

Further Tasks:

sequencing and further cloning of the complete constructs

2012-08-20

Preparative digestion of isolated CMV+AID without NES, with NLS+Kozak sequence+eGFP and AID without NES, with NLS+Kozak sequence with SpeI and PstI and TAL-Dom. with XbaI and PstI and Gelelectrophoresis

Investigators:

Tom S.

Time: 2012-08-20 8 am

Aim:

preparative digestion

Method:

Digestion with XbaI, PstI and SpeI, Gel electrophoresis

Results:

Gel extraction of digested samples from 20.08.2012

Investigators:

Chris

Method:

Gel extraction kit

Results:

CMV+AID without NES, with NLS+Kozak sequence+eGFP 2-1 (in pSB1C3)(digested with S and P): 236.2 ng/µL (96.6 nM)

CMV+AID without NES, with NLS+Kozak sequence+eGFP 2-2 (in pSB1C3)(digested with S and P): 37.0 ng/µL (15.1 nM)

AID without NES, with NLS+Kozak sequence 1-1-2 (in pSB1C3)(digested with S and P): 135.1 ng/µL (82.7)

AID without NES, with NLS+Kozak sequence 4-2-1 (in pSB1C3)(digested with S and P): 171.2 ng/µL (104.8 nM)

AID without NES, with NLS+Kozak sequence 4-2-2 (in pSB1C3)(digested with S and P): 96 ng/µL (58.7 nM)

AID for Fusion with TAL 2 (digested with X and P):51.1 ng/µL (136.6 nM)

AID for Fusion with TAL 3 (digested with X and P):35.8 ng/µL (95.5 nM)

Further Tasks:

Ligation

Ligation of CMV+AID without NES, with NLS+Kozak sequence+eGFP in pSB1C3 (S+P) & hGH poly A (X+P), AID without NES, with NLS+Kozak sequence in pSB1C3 (S+P) & hGH poly A (X+P), AID for Fusion with TAL (X+P) & pSB1C3 (X+P)

Investigators:

Chris

Materials:

T4 DNA-Ligase, T4-Ligase-buffer, water, samples

Method:

DNA Fragment ligation: according to the manual

sample preparation:

Fragment 1(BB)	Fragment 2(insert)	T4 DNA-Ligase	T4 DNA-Ligase Buffer	water
CMV+AID without NES, with NLS+Kozak sequence +eGFP 2-1 in pSB1C3 cut:S+P (96.6 nM)	hGH poly A cut: X+P (48.5 nM)
0.5 µL	2 µL	1 µL	1 µL	5.5 µL
CMV+AID without NES, with NLS+Kozak sequence +eGFP 2-2 in pSB1C3 cut:S+P (15.1 nM)	hGH poly A cut: X+P (48.5 nM)
7 µL	1 µL	1 µL	1 µL	0 µL
AID without NES, with NLS+Kozak sequence 4-2-1 in pSB1C3 cut S+P (82.7 nM)	hGH poly A cut: X+P (48.5 nM)
0.5 µL	2 µL	1 µL	1 µL	5.5 µL
AID without NES, with NLS+Kozak sequence 4-2-2 in pSB1C3 cut S+P (104.8 nM)	hGH poly A cut: X+P (48.5 nM)
0.5 µL	2 µL	1 µL	1 µL	5.5 µL
AID without NES, with NLS+Kozak sequence 1-1-2 in pSB1C3 cut S+P (58.7 nM)	AID without NES, with NLS+Kozak sequence 4(with P) cut: X+P (39.1 nM)
0.5 µL	2 µL	1 µL	1 µL	5.5 µL
AID for Fusion with TAL 2 cut: X+P (136.6 nM)	pSB1C3 cut: X+P (40.9 nM)
1 µL	1 µL	1 µL	1 µL	6 µL
AID for Fusion with TAL 3 cut: X+P (95.5 nM)	pSB1C3 cut: X+P (40.9 nM)
2 µL	1 µL	1 µL	1 µL	5 µL

incubation of samples for 1,5 h at room temperature

Results:

ligated plasmids ready for transformation

Further tasks:

Transformation

Topic: Transformation of ligated samples

Investigators: Chris

Materials:

samples (see above): ligated CMV+AID without NES, with NLS+Kozak sequence +eGFP +hGH poly A in pSB1C3, AID without NES, with NLS+Kozak sequence + hGH-polyA pSB1C3 , AID for Fusion with TAL in pSB1C3 (all chlR)

->1 plate per ligated sample, all in all 7 plates

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

Method:

Transformation via manual, 10 µl of ligation samples were used

Plate incubation start: 9.20 pm

Results:

ready mutants to pick clones

Further tasks:

picking clones

inoculation of pKMEF425bla, CMV in pSB1C3, CMV+wtAID+hGH-polyA in pSB1C3

Investigators: Chris

Time: 2012-08-16 7 pm

Materials:

LB medium

Chloramphenicol 25 mg/ ml stock solution

plates with cultures of pKMEF425bla (from 2012.08.16.)

cryo sotocks of CMV in pSB1C3 & CMV+wtAID+hGH-polyA in pSB1C3 Nr. 3

Method:

Inoculation of:

2 clones of pKMEF425bla in 5 ml LB,

1 tip of cryo stock from CMV in pSB1C3 (in 5 ml LB + Chloramph) and of CMV+wtAID+hGH-polyA in pSB1C3 Nr. 3 (in 50 ml LB + Chloramph)

(--> 4 cultures)

Further tasks:

Miniprep(pKMEF425bla, CMV in pSB1C3) and endotoxinfree mediprep (CMV+wtAID+hGH-polyA in pSB1C3) and cryo stocks

2012-08-21

Miniprep of pKMEF425bla & CMV in pSB1C3 CMV+wtAID+hGH-polyA in pSB1C3

Investigators: Chris

Materials:

Miniprep Kit

overnight cultures (5 mL) of pKMEF425bla and CMV in pSB1C3

Method:

according to manual

Results: concentrations:

CMV in pSB1C3 = 247.5 ng/µL

pKMEF425bla= 100 ng/µL

Further tasks:

preperative digestion of CMV with SpeI and PstI

test digestion of pKEF425bla with XbaI and AgeI

preperative digestion of pKEF425bla AscI and SfiI

endotoxin free mediprep CMV+wtAID+hGH-polyA in pSB1C3 for transfection in CHO

Investigators: Chris

Materials:

endotoxin free Mediprep Kit

overnight cultures (50 mL) of CMV+wtAID+hGH-polyA in pSB1C3

Method:

according to manual

Results: concentrations:

CMV+wtAID+hGH-polyA in pSB1C3(200 µL)=513.8 ng/µL

Further tasks:

Transfection into CHO cells

analytical digestion pKEF425bla (XbaI& AgeI) and preparative digestion of CMV-SpeI & PstI (with high and low DNA amount)

Investigators: Chris

Materials:

preparative CMV High:

25 µL CMV in pSB1C3 (247,5 ng/µL)

3 µL FD-Green Buffer

1 µL SpeI

1 µL PstI

preparative CMV Low:

6 µL CMV in pSB1C3 (247,5 ng/µL)

19 µL water

3 µL FD-Green Buffer

1 µL SpeI

1 µL PstI

test digestion pKEF425bla (NEB-Method)

2 µL pKMEF425bla (100 ng/µL)

23 µL water

3 µL NEB4 Buffer

1 µL XbaI

1µL AgeI

digestion incubated at 37°C for 2h 30 min

further tasks:

gel electrophoresis

gel electrophoresis (test digestion pKMEF425bla - XbaI & AgeI, preparative digestion CMV in pSB1C3- SpeI & PstI) and gel extraction of CMV in pSB1C3

concentrations after gel extraction:

pSB1C3+CMV high= 191.8 ng/µL

pSB1C3+CMV low= 55.4 ng/µL

inoculation of CMV+AID without NES, with NLS+Kozak sequence +eGFP+hGH, AID without NES, with NLS+Kozak sequence+hGH and TAL-AID in pSB1C3

Investigators: Chris, Tom S.

Time: 2012-08-16 7 pm

Materials:

LB medium

Chloramphenicol 25 mg/ml stock solution

plates with cultures of TAL-AID in pSB1C3 (from 2012.08.20.)

plates with cultures of CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH (from 2012.08.20.)

plates with cultures of AID without NES, with NLS+Kozak sequence+hGH (from 2012.08.20.)

Method:

Inoculation of:

2 clones per plate

(--> 14 cultures)

Further tasks:

Miniprep and cryo stocks

2012-08-22

Miniprep of overnight cultures of CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH, AID without NES, with NLS+Kozak sequence+hGH and TAL-AID in pSB1C3

Investigators:

Chris, Tom S.

Aim:

Miniprep of overnight cultures of CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH, AID without NES, with NLS+Kozak sequence+hGH and TAL-AID in pSB1C3

Method:

Miniprep Thermo Scientific: according to the manual

Results:

Concentrations

Sample	Concentration [ng/µL]
CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH 2-1-1	397.6
CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH 2-1-2	446.1
CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH 2-2-1	418.9
CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH 2-2-2	266.6
AID without NES, with NLS+Kozak sequence+hGH 1-2-2-1	319.3
AID without NES, with NLS+Kozak sequence+hGH 1-2-2-2	377.8
AID without NES, with NLS+Kozak sequence+hGH 4-2-1-1	415.0
AID without NES, with NLS+Kozak sequence+hGH 4-2-1-2	317.0
AID without NES, with NLS+Kozak sequence+hGH 4-2-2-1	307.0
AID without NES, with NLS+Kozak sequence+hGH 4-2-2-2	317.0
pBAD wt AID 4-2	211.6
TAL-AID in pSB1C3 2-1	291.1
TAL-AID in pSB1C3 2-2	305.4
TAL-AID in pSB1C3 3-1	460.5
TAL-AID in pSB1C3 3-2	292.2

Further Tasks:

Test digestion of all with PmlI, XbaI and PstI, and preparative digestion of AID without NES, with NLS+Kozak sequence+hGH with XbaI and PstI

test digestion of AID without NES, with NLS+Kozak sequence+hGH, CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH and TAL-Dom. in pSB1C3 with XbaI, PstI and PmlI, Gelelectrophoresis

Investigators:

Chris, Tom S.

Aim:

check which clone is the right one

Method:

Gel electrophoresis of all cut samples

Results:

we need to repeat the test digestion of CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH and AID without NES, with NLS+Kozak sequence+hGH;

Further Tasks:

sequencing and further cloning of the complete constructs; repeat the digestion

Preparative digestion of isolated AID without NES, with NLS+Kozak sequence+hGH-polyA with XbaI and PstI and gel extraction after gel electrophoresis

Investigators:

Chris, Tom S.

Time: 2012-08-22

Aim:

preparative digestion

Method:

Digestion with XbaI, PstI, SfiI and AscI, gel electrophoresis and gel extraction

Results:

AID without NES, with NLS+Kozak sequence+hGH-polyA 1-2-2-1 (in pSB1C3)(digested with X and P): 25.9 ng/µL -> 38.2 nM (MW: 678,7 kDa)

AID without NES, with NLS+Kozak sequence+hGH-polyA 1-2-2-2 (in pSB1C3)(digested with X and P): 18.2 ng/µL -> 26.8 nM

AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-1 (in pSB1C3)(digested with X and P): 39.4 ng/µL -> 58.1 nM

AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-2 (in pSB1C3)(digested with X and P): 38.7 ng/µL -> 57.0 nM

Further Tasks:

Ligation

2012-08-23

repetition of test digestion of AID without NES, with NLS+Kozak sequence+hGH-polyA and CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA+eGFP with XbaI, PstI and PmlI, gel electrophoresis

Investigators:

Chris

Aim:

check which clone is the right one

Method:

Gel electrophoresis of all digested samples

Results:

Further Tasks:

sequencing and further cloning of the complete constructs

Ligation of AID without NES, with NLS+Kozak sequence+hGH-polyA (X+P) with pSB1C3+CMV (S+P)

Investigators:

Chris

Materials:

T4 DNA-Ligase, T4-Ligase-buffer, water, samples

Method:

DNA Fragment ligation: according to the manual

sample preparation:

Fragment 1(BB)	Fragment 2(insert)	T4 DNA-Ligase	T4 DNA-Ligase Buffer	water
pSB1C3+CMV S+P low(32 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 1-2-2-1 cut: X+P (38.2 nM)
1 µL	3 µL	1 µL	1 µL	4 µL
pSB1C3+CMV S+P high(191 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 1-2-2-1 cut: X+P (38.2 nM)
0.5 µL	7.5 µL	1 µL	1 µL	0 µL
pSB1C3+CMV S+P low(32 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 1-2-2-2 cut: X+P (26.8 nM)
1 µL	4 µL	1 µL	1 µL	3 µL
pSB1C3+CMV S+P high(191 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 1-2-2-2 cut: X+P (26.8 nM)
0.5 µL	7.5 µL	1 µL	1 µL	0 µL
pSB1C3+CMV S+P low(32 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-1 cut: X+P (58.1 nM)
2 µL	4 µL	1 µL	1 µL	2 µL
pSB1C3+CMV S+P high(191 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-1 cut: X+P (58.1 nM)
0.5 µL	6 µL	1 µL	1 µL	1.5 µL
pSB1C3+CMV S+P low(32 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-2 cut: X+P (57 nM)
2 µL	4 µL	1 µL	1 µL	2 µL
pSB1C3+CMV S+P high(191 nM)	AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-2 cut: X+P (57 nM)
0.5 µL	6 µL	1 µL	1 µL	1.5 µL

incubation of samples for 1,5 h at room temperature

Results:

ligated plasmids for transformation

Further tasks:

Transformation

Topic: Transformation of ligated samples

Investigators: Chris

Materials:

samples (see above): AID without NES, with NLS+Kozak sequence+hGH-polyA (X+P) with pSB1C3+CMV (S+P) (chlR)

-> 1 plates per ligated sample, 8 plates

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

Method:

Transformation via manual, 10 µl of ligation samples were used

Plate incubation start: 6 pm

Results:

ready mutants to pick clones

Further tasks:

picking clones

2012-08-24

send the DNA to sequencing

Investigators:

Tom S.

mod.AID = AID without NES, with NLS+Kozak sequence

sample	GATC number	Seq. Primer	GATC number
mod. AID 1-1-2 Forward	II3282	pSB1C3 Forward	919656
mod. AID 1-1-2 Reverse	II3294	pSB1C3 Reverse	919657
mod. AID 4-2-1 Forward	II3283	pSB1C3 Forward	919656
mod. AID 4-2-1 Reverse	II3295	pSB1C3 Reverse	919657
mod. AID 4-2-2 Forward	II3284	pSB1C3 Forward	919656
mod. AID 4-2-2 Reverse	II3296	pSB1C3 Reverse	919657
mod. AID+eGFP 3 Forward	II3285	pSB1C3 Forward	919656
mod. AID+eGFP 3 Reverse	II3297	pSB1C3 Reverse	919657
CMV+mod. AID+eGFP+hGH 2-1-1 Forward	II3286	pSB1C3 Forward	919658
CMV+mod. AID+eGFP+hGH 2-1-1 Reverse	II3298	pSB1C3 Reverse	919659
CMV+mod. AID+eGFP+hGH 2-1-1 AID	II3299	AID-C-Term Forward	919660
CMV+mod. AID+eGFP+hGH 2-1-1 eGFP	II3300	eGFP-N-Term Reverse	919661
CMV+mod. AID+eGFP+hGH 2-1-2 Forward	II3287	pSB1C3 Forward	919658
CMV+mod. AID+eGFP+hGH 2-1-2 Reverse	II3301	pSB1C3 Reverse	919659
CMV+mod. AID+eGFP+hGH 2-1-2 AID	II3302	AID-C-Term Forward	919660
CMV+mod. AID+eGFP+hGH 2-1-2 eGFP	II3303	eGFP-N-Term Reverse	919661
CMV+mod. AID+eGFP+hGH 2-2-1 Forward	II3288	pSB1C3 Forward	919658
CMV+mod. AID+eGFP+hGH 2-2-1 Reverse	II3304	pSB1C3 Reverse	919659
CMV+mod. AID+eGFP+hGH 2-2-1 AID	II3305	AID-C-Term Forward	919660
CMV+mod. AID+eGFP+hGH 2-2-1 eGFP	II3306	eGFP-N-Term Reverse	919661
CMV+mod. AID+eGFP+hGH 2-2-2 Forward	II3289	pSB1C3 Forward	919658
CMV+mod. AID+eGFP+hGH 2-2-2 Reverse	II3307	pSB1C3 Reverse	919659
CMV+mod. AID+eGFP+hGH 2-2-2 AID	II3308	AID-C-Term Forward	919660
CMV+mod. AID+eGFP+hGH 2-2-2 eGFP	II3309	eGFP-N-Term Reverse	919661
TAL-Dom. 2-1 Forward	II3290	pSB1C3 Forward	919662
TAL-Dom. 2-1 Reverse	II3310	pSB1C3 Reverse	919663
TAL-Dom. 2-2 Forward	II3291	pSB1C3 Forward	919662
TAL-Dom. 2-2 Reverse	II3311	pSB1C3 Reverse	919663
TAL-Dom. 3-1 Forward	II3292	pSB1C3 Forward	919662
TAL-Dom. 3-1 Reverse	II3312	pSB1C3 Reverse	919663
TAL-Dom. 3-2 Forward	II3293	pSB1C3 Forward	919662
TAL-Dom. 3-2 Reverse	II3313	pSB1C3 Reverse	919663

Preparative digestion of pAK 100 and scFV with AscI and SfiI

Investigators:

Rico

Aim:

digest pAK 100 and scFV into fragments

Method:

3 µL NEB4 buffer, 1 µL AscI, 25 µL DNA, water

4 h -> 37 °C

ad 1 µL SfiI, 4 h -> 50 °C

2012-08-27

gel electrophoresis of pAK100 (SfiI & AscI) & pKMEF425bla (AscI+SfiI) and gel extraction of pKMEF425bla (AscI+SfiI)

Investigators:

Basia

Results:

final concentration pKMEF425bla (AscI+SfiI):3.4 ng/µL

send the DNA to sequencing

Investigators:

Rico, Chris

</tr>

sample	GATC number	Seq. Primer	GATC number
pBad+WtAID 4-2	II3321	pBAD-FP	10199242
pBad+WtAID 4-2	II3322	pTrcHis-RP	10199243
pBad+WtAID 4-1	II3323	pBAD-FP	10199244
pBad+WtAID 4-1	II3324	pTrcHis-RP	10199245
pBad+WtAID 1-1	II3325	pBAD-FP	10199246
pBad+WtAID 1-1	II3326	pTrcHis-RP	10199247

inoculation of cryo stocks of pAK100 and pKMEF425bla; CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA

Investigators: Chris, Rico

Time: 2012-08-16 7 pm

Materials:

LB medium

Chloramphenicol 25 mg/ ml stock solution

plates with cultures CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA: 4-2-2-1 L, 4-2-2-1 H, 4-2-2-2 L, 4-2-2-2 H, 1-2-2-1 L, 1-2-2-1 H, 1-2-2-2 L, 1-2-2-2 H

cryostocks of pAK100 and pKMEF425bla

Method:

Inoculation of:

1 clones per plate

(--> 8 cultures)

Further tasks:

Miniprep and cryo stocks

2012-08-28

Miniprep and cryostocks of overnight cultures

Investigators: Rico

Aim: purification of the CMV+AID without NES, with NLS+Kozak sequence+hGH in pSB1C3 construct

Method:

Miniprep Thermo Scientific

Results:

pAK 100 = 414.8 ng/µL

1-2-2-1-H = 311,3 ng/µL

1-2-2-1-L = 249,4 ng/µL

1-2-2-2-H = 303,5 ng/µL

1-2-2-2-L = 230,9 ng/µL

4-2-2-1-H = 259,3 ng/µL

4-2-2-1-L = 288,3 ng/µL

4-2-2-2-H = 513,8 ng/µL

4-2-2-2-L = 325,7 ng/µL

Further Tasks:

Test digestion of these samples with PmlI, XpaI and PstI

Test digestion of CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA with PmlI, XpaI and PstI

Investigators: Basia, Rico

Aim: Test digestion of CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA construct with PmlI, XpaI and PstI

Method:

1 µL DNA, 1 µL PmlI, 1 µL PstI, 1 µL XbaI, 3 µL FD Buffer, 23 µL water

incubation for 1,5 h at 37 °C

Results:

300px

Checking the sequencing data

Investigators: Tom S.

Aim: choose the right constructs

Results:

AID without NES, with NLS+Kozak sequence in pSB1C3 1-1-2 -> construct not the right one

AID without NES, with NLS+Kozak sequence in pSB1C3 4-2-1 -> mutation of the 4th nt in the Kozak sequence (impact can't estimate)

AID without NES, with NLS+Kozak sequence in pSB1C3 4-2-2 -> is good

TAL-Dom. in pSB1C -> all ok

CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH -> the flanking sequences are good, but the middle primers were wrong and must be repeated

2012-08-29

Preparative digestion of pAK 100 with AscI and SfiI

Investigators:

Rico

Aim:

Preparative digestion of pAK 100 with AscI and SfiI for Phage Display

Method:

10 µL DNA, 15 µL water, 3 µL 10x FD Buffer, 1 µL AscI 6 h @ 37 °C

add 1 µL SfiI 5 h @ 50 °C

Further Tasks:

gel electrophoresis

Test digestion of CMV+AID without NES, with NLS+Kozak sequence+hGH construct with PmlI, PstI and XbaI

Investigators:

Basia, Rico

Aim:

Test digestion of CMV+AID without NES, with NLS+Kozak sequence+hGH 4-2-2-2 L, 4-2-2-2 H, 4-2-2-1 L, 4-2-2-1 H

Method:

0.5 µL DNA, 0.5 µL PmlI, 0.5 µL PstI, 0.5 µL XbaI, 1 µL 10x FD Buffer, 7 µL water

Results:

send the DNA to sequencing

Investigators:

Tom S.

sample	GATC number	Seq. Primer	GATC number
CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA 2-1-1	II3331	AID-C-Term. Forward	919664
CMV+AID without NES, with NLS+Kozak sequence+eGFP-hGH-polyA 2-1-1	II3332	eGFP-N-Term. Reverse	919665
CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-1-L	II3333	pSB1C3 Forward	919666
CMV+AID without NES, with NLS+Kozak sequence+hGH 4-2-2-1-L	II3334	pSB1C3 Reverse	919667

2012-08-30

Endotoxin-free mediprep of CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA construct

Investigators:

Rico, Tom,

Method:

Midiprep

Result:

CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA 2-1-2 = 1546.7 ng/µL

CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA 2-2-1 = 1115.0 ng/µL

CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA 2-1-1 = 1358.4 ng/µL

CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA 2-2-2 = 1518.9 ng/µL

POG 4 = 1518.9 ng/µL

Clone 4 = 1239.0 ng/µL

Further Tasks:

construct is ready for transfection

Miniprep of YFP-construct from transfected CHO-Cells

Investigators:

Rico, Tom

Aim:

Miniprep of YFP-construct from transfected CHO cells to proof whether it is possible to transform E. coli with the purified construct for sequencing

Method:

harvesting of the CHO-cells with 0.3 mL Trypsin/EDTA and 0.5 mL medium Ham's F-12 (2.5 * 10^5 cells were seeded 3 day before)

Miniprep of cells

Elution with 20 µL

Results:

1 µg DNA with 1 µL PEI = 33.3 ng/µL

1 µg DNA with 1.5 µL PEI = 55.2 ng/µL

1 µg DNA with 2 µL PEI = 30.9 ng/µL

1 µg DNA with 2.5 µL PEI = 29.4 ng/µL

1 µg DNA with 3 µL PEI = 40.2 ng/µL

1 µg DNA with 3.5 µL PEI = 63.4 ng/µL

Topic: transformation of Venus vector

Investigators: Basia

Time: 2012-08-30

Materials:

Bunsen Burner, Agar Plates with chloramphenicol

icebox

competent E. coli cells (XL 1 Blue)

Venus plasmid - sample 1

Method:

Transformation via manual, 2 µl of Venus plasmid was used

Plate incubation start: 7:30 pm

Results:

ready mutants to pick clones

Further tasks:

picking clones

Preparative digestion of pAK 100 with AscI and SfiI and gel extraction

Investigators:

Chris, Rico

Results:

c(pAK 100 cut:AscI and SfiI) from left lane:9 ng/µL

c(pAK 100 cut:AscI and SfiI) from right lane: 19 ng/µL

because the fragments where not separated well we repeated the digestion (simultanously going on with ligation & transformation)

Ligation & transformation of pAK 100(AscI and SfiI) & scFV425bla(AscI and SfiI)

Ligation:

Fragment 1(BB)	Fragment 2(insert)	T4 DNA-Ligase	T4 DNA-Ligase Buffer	water
pAK 100-left lane(9 ng/µL)	425bla (3 ng/µL)
1/3 µL	3 µL	1 µL	1 µL	4,6 µL
pAK 100-right lane(50% diluted->9.5 ng/µL)	425bla (3 ng/µL)
1 µL	3 µL	1 µL	1 µL	6 µL
pAK 100-right lane(50% diluted->9.5 ng/µL)	425bla (3 ng/µL)
0.1 µL	4.5 µL	1 µL	1 µL	3 µL

Transformation:

Transformation via protocol, 10 µL of ligated samples were used

Results:
plates with transformed E. coli

Further Tasks:
picking clones

repetition of preparative digestion of pAK 100 with AscI and SfiI

sample:

2.4 µL(~1000 ng) pAK 100

3 µL 10x NEB 4 Buffer

2 µL AscI

22.6 µL water

incubation at 37 °C for 5 h and heat inactivation for 20 min at 65°C, freeze at - 20°C

Further Tasks:
digestion with SfiI

Checking the sequencing data

Investigators: Chris, Tom S.

Aim: choose the right constructs

Results:

CMV+AID without NES, with NLS+Kozak sequence+eGFP+hGH-polyA 2-1-1 -> is good

CMV+AID without NES, with NLS+Kozak sequence+hGH-polyA 4-2-2-1-L -> is good

pBAD+WT AID 1-1 -> 3 bp in front of start codon and direct after the stop codon are mutations

pBAD+WT AID 4-1 -> 3 bp in front of start codon and direct after the stop codon are mutations

pBAD+WT AID 4-2 -> 3 bp in front of start codon and direct after the stop codon are mutations

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 CaCl2.

2012-08-31

Topic: preparation of competent XL1 Blue E. coli

Investigators:Basia

Aim:

get competent E. coli Xl1 Blue

Materials:

CaCl2, overnight culture of E. coli XL1 Blue, Eppendorf tubes

Method:

Competent E. coli -> Standard protocols

Results:

ca. 80 (100 µl) Stocks frozen competent E. coli XL1 Blue

location: 2 paper boxes with competent E. coli Xl1 Blue in -80°C Freezer (middle shelf, most right)

Further tasks:

testing ability for transformation of competent cells

Topic: picking clones &inoculation in 5 mL LB of retransformed Venus out of CHO cells

Investigators: Tom S.

Method: picking clone (and inoculation in 5 ml LB medium + 5µl chloramphenicol stock, shaking over night at 37°C, 300 rpm, approx. 16 hours

Further tasks:

Miniprep

Topic: picking clones &inoculation in 5 mL LB of pAK 100(AscI and SfiI) & scFV425bla(AscI and SfiI)

Investigators: Chris

Method: picking clones (and inoculation in 5 ml LB medium + 5µl chloramphenicol stock, shaking over night at 37°C, 300 rpm, approx. 16 hours

Further tasks:

Miniprep

Antibody

2012-08-02

Planning the antibody construct with new antibody (GCN4) and optimizing the old ones

Investigators: Maria

Time: 2012-08-02 9- 15 pm

Materials: Geneious

Results: optimized constructs with scFv 425bla, scFv425-72000, scFv GCN4

miniprep of ligated pcDNA5-FRT-scFv-TEV-TMD-EYFP genecostruct from 2012-07-31

Investigators: Sascha

Materials:

Miniprep Kit

o.n. cultures of 2 clones from each ligation (1:2, 1:3, 1:4)

Method:

according to manual

Results: concentration measurement of preped ligated geneconstrcuts

clone 1 from 1:2 ligation=

clone 2 from 1:2 ligation=

clone 1 from 1:3 ligation=

clone 2 from 1:3 ligation=

clone 1 from 1:4 ligation=

clone 2 from 1:4 ligation=

further tasks:

analytical digestion

analytical digestion of ligated pcDNA5-FRT-scFv-TEV-TMD-EYFP genecostruct from 2012-07-31

Investigators: Sascha

Materials:

all 6 preped ligated pcDNA5-FRT-scFv-TEV-TMD-EYFP genecostruct from 2012-07-31

FastDigest NheI

10x FD Green Buffer

steril water

Method:

10µl mix: 1µl NheI, 1µl 10x FD Green Buffer, 1µl of each clone (approximately 850ng DNA), steril water ad to 10µl

digestion incubated at 37°C for 30´

further tasks:

gelelectrophoresis

Topic: gelelectrophoresis of analytical digested pcDNA5-FRT-scFv-TEV-TMD-EYFP genecostruct from 2012-07-31

Investigators: Sascha

Aim: checking plasmid-size after ligation of pcDNA5-FRT-scFv-TEV-TMD-EYFP in 1% agarosegel

Materials:

agarose

1xTAE-buffer

10xFD Green Buffer

Method:

1% agarosegel, 90ml

70 min at 115V

Results:

ligation failed

Further Tasks:

amplifying new scFv-TEV-TMD-EYFP geneconstruct

preparative digestion of pcDNA5-FRT and scFv-TEV-TMD-EYFP geneconstruct

ligation of pcDNA5-FRT and scFv-TEV-TMD-EYFP geneconstruct

2012-08-03

Reviewing old antibody constructs and planning construct with new anti-GFP nanobody

Investigators: Maria

Time: 2012-08-03 10 - 17 pm

Materials: Geneious

Results: optimized constructs with anti-GFP nanobody and mcherry

amplifying of scFv-TEV-TMD-EYFP geneconstruct

Investigators: Sascha

Aim: amplifying of scFv-TEV-TMD-EYFP geneconstruct

Materials:

final assembeld scFv-TEV-TMD-EYFP geneconstruct from 2012-07-28

P7_Gesamtanfang (forward-primer)

P8_Gesamtende (reverse-primer)

Phusion -polymerase, dNTPs (10mM), Phusion HF-Buffer

Method:

pcr-mix: 10µl HF-Buffer; 1µl dNTPs(10mM); 2µl of final assembeled scFv-TEV-TMD-EYFP geneconstruct from 2012-07-28;2,5µl of P7_Gesamtanfang (forward-primer); 2,5µl of P8_Gesamtende (reverse-primer); nuclease-free water ad to 50µl; 0,5µl Phusion-polymerase

PCR-prgram with end-primers: initial Denat. 40´´ at 98°C; Denat 12´´ at 98°C; Annealing 15´´ at 57°C; Elongation 27´´ at 72°C; final-Elongation 10´at 72°C; 32 cycles

further Tasks:

PCR-Clean-Up

analytical gelelectrophoresis of cleaned and amplifyied scFv-TEV-TMD-EYFP genecostruct

digestion of cleaned and amplifyied scFv-TEV-TMD-EYFP geneconstruct

PCR-Clean-Up of scFv-TEV-TMD-EYFP geneconstruct

Investigators: Sascha

Aim: cleaning of scFv-TEV-TMD-EYFP genecostruct

Materials:

PCR-Clean-Up Kit

Method:

according to manual

Results: concentration measurement via nanodrop

concentration of cleaned scFv-TEV-TMD-EYFP genecostruct=

analytical gelelectrophoresis of cleaned and amplifyied scFv-TEV-TMD-EYFP geneconstruct

Investigators: Sascha

Aim: analytical gelelectrophoresis to check size of amplifyied scFv-TEV-TMD-EYFP geneconstruct in 1% agarosegel

Materials:

agarose

1xTAE-buffer

10xFD Green Buffer

Method:

1µl 10x FD Green, 1µl of amplifyied scFv-TEV-TMD-EYFP geneconstruct, water add to 10µl

1% agarosegel, 90ml

70 min at 120V

Results:

amplification worked --> DNA-bond at 1701bp

Further Tasks:

preparative digestion of pcDNA5-FRT and scFv-TEV-TMD-EYFPv geneconstruct

ligation of pcDNA5-FRT and scFv-TEV-TMD-EYFPv geneconstruct

preparative digestion of pcDNA5-FRT from

Investigators: Sascha

Aim: digestion of pcDNA5-FRT with NheI and ApaI to generate sticky ends for ligation with scFv-TEV-TMD-EYFPv geneconstruct

Materials:

Fast Digest NheI

Fast Digest ApaI

10x FD Green Buffer

pcDNA5-FRT(gut) from

steril water

Method:

2,5µl pcDNA5-FRT/gut (729,ng/µl) --> 1822ng

2µl NheI

2µl ApaI

3µl 10x FD Green Buffer

water add to 30µl

digestion for 2,5h at 37°C

Further Tasks:

dephosphorylation

gelelectrophoresis

gelextraction

dephosphorylation of pcDNA5FRT digested with NheI and ApaI

Investigators: Sascha

Aim:dephosphorylation of digestd pcDNA5-FRT to prevent religation

Materials:

Antarctic Phosphatase

10x Antarctic Phosphatase Reaction Buffer

digested pcDNA5-FRT

Method:

3,3µl 10x Antarctic Phosphatase Reaction Buffer

1µl Antarctic Phosphatase

incubation for 30min at 37°C to dephosphorylate 5´-ends of pcDNA5-FRT

heat inactivation of Phosphatase for 5min at 65°C

store at -20°C

Further Tasks:

gelelectrophoresis

gelextraction

digestion of scFv-TEV-TMD-EYFP geneconstruct

2012-08-05

preparative digestion of scFv-TEV-TMD-EYFPv geneconstruct

Investigators: Kerstin

Aim: digestion of scFv-TEV-TMD-EYFPv geneconstructwith NheI and ApaI to generate sticky ends for ligation with digested pcDNA5-FRT

Materials:

Fast Digest NheI

Fast Digest ApaI

10x FD Green Buffer

scFv-TEV-TMD-EYFPv geneconstruct

steril water

Method:

26µl final assembled scFv-TEV-TMD-EYFP geneconstruct(76,4µng/µl) --> 1986,4ng

1µl NheI

1µl ApaI

3,1µl 10x FD Green Buffer

water add to 31µl

digestion for 2,5h at 37°C

heat inactivation of NheI and ApaI --> 5min at 65°C

store at -20°C

Further Tasks:

gelelectrophoresis

gelextraction

ligation with digested pcDNA5-FRT

2012-08-06

Reviewing new construct with anti-GFP nanobody and contacting IDT and GeneArt

Investigators: Maria

Time: 2012-08-06 12 - 14 pm

Materials: Geneious

Results: final construct with anti-GFP nanobody and mcherry

preparative gelelectrophoresis of digested scFv-TEV-TMD-EYFP geneconstruct and digested + dephosporylated pcDNA5-FRT

Investigators: Sascha

Aim: preparative gelelectrophoresis to separate digested scFv-TEV-TMD-EYFP geneconstruct and pcDNA5-FRT in 1% agarosegel

Materials:

agarose

1xTAE-buffer

digested DNA

Method:

30µl mix of digested and dephosphorylated pcDNA5-FRT

31µl mix of digested scFv-TEV-TMD-EYFP geneconstruct

1% agarosegel, 100ml

90 min at 95V

Results:

Further Tasks:

gelextraction

ligation

gelextracton of digested scFv-TEV-TMD-EYFP geneconstruct and digested + dephosporylated pcDNA5-FRT out of 1% agarosegel

Investigators: Sascha

Aim: extract of digested scFv-TEV-TMD-EYFP geneconstruct and pcDNA5-FRT

Materials:

Gel-Clean-Up Kit

Method:

according to manual

failure at elution step

Further Tasks:

pcr-Clean-up of digested scFv-TEV-TMD-EYFP geneconstruct and pcDNA5-FRT

PCR-Clean-Up of digested scFv-TEV-TMD-EYFP geneconstruct and digested + dephosporylated pcDNA5-FRT after gelextraction

Investigators: Sascha

Aim: cleaning and eluting of digested scFv-TEV-TMD-EYFP geneconstruct and pcDNA5-FRT

Materials:

PCR/Gel-Clean-Up Kit

Method:

according to manual

Results: concentration measurement using nanodrop

concentration of cleaned-up pcDNA5-FRT= 8,4ng/µl

concentration of celaned-up scFv-TEV-TMD-EYFP geneconstruct= 10,4ng/µl

Further Tasks:

ligation of pcDNA5-FRT and scFv-TEV-TMD-EYFP geneconstruct

ligation of digested scFv-TEV-TMD-EYFP geneconstruct and digested + dephosporylated pcDNA5-FRT

Investigators: Sascha

Aim: ligation of digested scFv-TEV-TMD-EYFP geneconstruct and pcDNA5-FRT

Materials:

ligation calculator: http://www.insilico.uni-duesseldorf.de/Lig_Input.html

T4 DNA ligae

ligase buffer

digested scFv-TEV-TMD-EYFP geneconstruct

digested + dephosporylated pcDNA5-FRT

Method: ligation-ratio--> 1:3

1µl T4 DNA ligase

2,5µl T4 DNA-ligase buffer

11,9µl digested + dephosporylated pcDNA5-FRT (8,4ng/µl) --> 100ng

9,67µl digested scFv-TEV-TMD-EYFP geneconstruct (10,4ng/µl) --> 100,66ng

no religation-copntrol

incubation for 1h at RT

Further Tasks:

transformation of ligation mix into XL1-blue competente E. coli cells

Transformation of ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP into XL1-blue competente E. coli cells

Investigators: Sascha

Materials:

Bunsen Burner, Agar Plate with Ampicilin, 37 °C heater, centrifuge,

ligase sample

icebox

competent E. coli cells (XL 1)

Method:

Transformation via manual

50µl of resuspended cell-suspension were plated on a LB-Amp-plate

incubation o.n. at 37°C

Further Tasks:

picking clones

2012-08-07

Preparing GeneArt order of nanobody construct

Investigators: Maria

Time: 2012-08-07 09 - 11 am

Materials: Geneious

Results: Analysis and quotation by GeneArt (24h processing time)

colony-pcr of ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Investigators: Sascha

AIM: identifying correct ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Materials:

Taq DNA-polymerase 1kb/1min

10x Taq DNA-polymerase buffer

dNTPs (10mM)

forward primer in C-terminus of CMV of pcDNA5-FRT-backbone: fp_qRT-CMV_rev (Tm=55°C)

reverse primer in C-terminus in EYFP: ps_mcherry_rev_BamHI (Tm= 59,31°C)

Thermocycler

Methods:

20µl colony-pcr mix for one clone: 2µl 10x Standard Taq-DNA-Polymerase Buffer; 0,4µl dNTPs (10mM), 0,4µl fp_qRT-CMV_rev(10µM), 0,4µl ps_mcherry_rev_BamHI (10µM); 0,1µl Taq-DNA-Polymerase; nuclease-free water add to 20µl

colony-pcr mix were adapted to 26 clones

after transferation of clone into 20µl colony-pcr mix, the rest of clone at the tip was plated on a LB-AMP-plate

LB-AMP-plate were incubated o.n. at 37°C

negative control: clon #24 from relagtion-control (ligation-ratio 1:0) from 2012-07-31

program of colony-pcr: initial Denaturation:5´at 95°C; Denaturation: 25´´ at 95°C, Annealing: 25´´ at 51°C; Elongation: 114´´ (1845bp) at 68°C; final Elongation: 5´at 86°C; 30 cycles; store at 8°C

further Taks:

gelelectrophoresis

picking positiv clones for o.n.-culture

gelelectrophoresis of colony-pcr

Investigators: Sascha

AIM: identifying correct ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Materials:

1 100 ml 1%agarosegel with 20 slots

1 90 ml 1% agarosegel with 9 slots

1x TAE-buffer

agarose

Method:

90 ml 1% agarosegel for 80 min at 110V

100 ml 1% agarosegel for 100 min at 110V

Results:

(positive) clones 3, 4, 5, 8, 21, 23 were chosen for o.n.-culture because of desired DNA-bond at appropriate 1845bp

further Tasks:

o.n.-culture of positive clones

o.n.-culture of positive ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Investigators: Tom S.

Materials:

8x 15 ml Falcons

LB-Medium

Ampicilin

bunsen burner

Method:

2x o.n.-culture of clones 8 and 23

1x o.n.-culture of clones 3,4,5,21

each o.n.-culture 5ml LB-Medium + 5µl Ampicilin

incubation o.n. at 37°C

further Tasks:

miniprep of o.n.-cultures

2012-08-08

Ordering nanobody construct from GeneArt

Investigators: Maria

Time: 2012-08-08 10 - 11 am

Materials: Geneious

Results: Final nanobody construct, estimated producing time aprox. 16 days

miniprep of o.n.-culture of positive ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Investigators: Stefan

Materials:

Miniprep-Kit

Method:

according to manual

Results: concentration measurement via nanodrop

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone3=

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone4=

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone5=

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone8=

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone21=

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone23=

further Tasks:

looking for primer for GATC-sequencing of positive pcDNA5-FRT_scFv-TEV-TMD-EYFP_clones

2012-08-10

preparing ligated pcdna5frt-scfv-tev-tmd-eyfp-construct for ordering sequencing by GATC

Investigators: Sascha

Materials:

dilute DNA (clons 3,8,21) according to GATC requirements
Geneious
Using GATC standard-primer pcdna3.1FP.1 and pcdna3.1RP.1

Further Tasks:

analysis of sequencing results

2012-08-13

Cell culture

Investigators: Kerstin

Time: 2012-08-13 3 pm

Materials: culture flasks (75cm²), 6-Well Plates, Trypsin/EDTA, PBS, Ham's F12 Medium

Method:

passage of CHO Flp-In cells, 1:10 dilution

Preparation for Transfection: seeding of CHO Flp-In cells in 6-Well plates (1*10^5, 1,5*10^5, 2*10^5,..., 3,5*10^5 cells/well)

Incubation for 2 days

2012-08-15

Transfection of CHO cells with WildTyp AID and CMV_mVenus_construct

Investigators: Kerstin, Stefan

Time: 2012-08-15 11 am

Materials: cells in 6-Well Plates, DNA WildTyp AID, CMV_mVenus_construct, Opti-MEM, PEI Transfection Reagent

Method:

wells with 80% confluence were chosen for transfection (2,5*10^5cells/well seeded on 13.08.12)

protocol 1:

4,8fold PEI transfection reagent according to DNA amount

2,5µg CMV_mVenus_construct DNA + 2,5µg WildTYp AID DNA, add to 200µl with Opti-MEM

24µl PEI + 176µl Opti-MEM

vortex 3 times for 10 sec

incubate for 2 min

mix solutions (always add PEI mix to DNA mix)

incubation for 15 min

spread on cells and mix gently

protocol 2:

2,5fold PEI transfection reagent according to DNA amount

1µg CMV_mVenus_construct DNA + 1µg WildTYp AID DNA, add to 200µl with Opti-MEM

5µl PEI + 195µl Opti-MEM

vortex 3 times for 10 sec

incubate for 2 min

mix solutions (always add PEI mix to DNA mix)

incubation for 15 min

spread on cells and mix gently

Results:

fluorescence image of protocol 1 treated cells

File:UP 12 Transfection 16.08 prot1.tiff

2012-08-15

Retransformation with scFv, transmembrane domain and YFP

Investigators: Kerstin/Stefan

Aim: Retransformation with scFv anti-EGFR

Date/Time: 15th August 2012, 2:30 – 4:30 pm

Materials: competent E. coli cells XL1 Blue, scFv anti-EGFR, 42°/37° C shaker, centrifuge

Method: see transformation protocol

Results: colonies on Chloramphenicol plates with scFv

Further tasks: picking clones and overnight culture (16th August)

2012-08-17

extension-pcr of scFv-TEV-TMD-EYFP geneconstruct

Investigators: Kerstin/Stefan

Materials:
scFv anti-EGFR, BBa-KI57000 with transmembrane domain; BBa_E0030 with EYFP

P1_Signalp_N-term

P2_Signalp_C-term

P3_TMD-N-term

P4_TMD-C-term/N-YFP

P5_YFP-N-term/TMD-C-term

P6_YFP-C-term

Phusion -polymerase, dNTPs (10mM), Phusion HF-Buffer

Method:

50µl PCR mix of each plasmid= 10µl HF-Buffer; 1µl dNTPs(10mM); forward and reverse primer each 2,5µl (10µM); template DNA: 1µl of 200ng/µl scFv--> 200ng, 5µl of BBa_E0030 with EYFP (46,3ng/µl--> ca. 230ng),5µl of BBa-KI57000 with transmembrane domain (43,9ng/µl --> ca. 230ng); Phusion DNA polymerase 0,5µl; nclease-free water ad to 50µl

PCR-program: initial Denat. 40´´ at 98°C; Denat 12´´ at 98°C; Annealing 15´´ at 55°C; Elongation 11´´ at 72°C; final-Elongation 10´at 72°C; 30 cycles

Further Tasks:

gelelectrophoresis

gelelectrophoresis of extended genes after extension-pcr

Investigators: Kerstin/Stefan

Aim: separation of extended genes scFv-TEV, TEV-TMD, EYFP in 1% agarosegel

Materials:

agarose

1xTAE-buffer

10xFD Green Buffer

extended genes

Method:

1% agarosegel

70 min at 105V

Results:

Further Tasks:

gelextraction

gelextraction of extended genes after extension-pcr

Investigators: Kerstin/Stefan

Aim: gelextraction of extended genes(scFv-TEV, TEV-TMD, EYFP) out of 1% agarosegel

Method:

DNA extracted according to the manual

Further Tasks:

assembly-pcr

assembly-pcr with extended genes to scFv-TEV-TMD-EYFP geneconstruct

Investigators: Kerstin/Stefan

Aim: assembly-pcr with extended genes to scFv-TEV-TMD-EYFP geneconstruct

Materials:
scFv anti-EGFR, BBa-KI57000 with transmembrane domain; BBa_E0030 with EYFP

P7_Gesamtanfang (forward-primer)

P8_Gesamtende (reverse-primer)

Phusion -polymerase, dNTPs (10mM), Phusion HF-Buffer

Method:

assembly-pcr-mix--> 3:1:3 10µl HF-Buffer; 1µl dNTPs(10mM); extended TMD (1ng), extended EYFP (2,75ng), extended scFv (2,75ng); nuclease-free water ad to 45µl; 0,5µl Phusion-polymerase

PCR-program to let the extended genes prime each other: initial Denat. 40´´ at 95°C; Denat 12´´ at 95°C; Annealing 15´´ at 68°C; Elongation 27´´ at 72°C; final-Elongation 10´at 72°C; 35 cycles

after annealing of extended genes to each other 2,5µl of primer forw_P7_Gesamtanfang and primer rev_P8_Gesamtende were added

PCR-prgram with end-primers: initial Denat. 40´´ at 95°C; Denat 12´´ at 95°C; Annealing 15´´ at 55°C; Elongation 27´´ at 72°C; final-Elongation 10´at 72°C; 30 cycles

Gelelectrophoresis at 105V for 75`

Results:

extraction of DNA (1701bp)

Further Tasks:

Amplification of assembly pcr-Product

2012-08-18

amplifying of scFv-TEV-TMD-EYFP geneconstruct

Investigators: Kerstin/Stefan

Aim: amplifying of scFv-TEV-TMD-EYFP geneconstruct

Materials:

final assembeld scFv-TEV-TMD-EYFP geneconstruct from 2012-07-28

P7_Gesamtanfang (forward-primer)

P8_Gesamtende (reverse-primer)

Phusion -polymerase, dNTPs (10mM), Phusion HF-Buffer

Method:

pcr-mix: 10µl HF-Buffer; 1µl dNTPs(10mM); 2µl of final assembeled scFv-TEV-TMD-EYFP geneconstruct from 2012-07-28;2,5µl of P7_Gesamtanfang (forward-primer); 2,5µl of P8_Gesamtende (reverse-primer); nuclease-free water ad to 50µl; 0,5µl Phusion-polymerase

PCR-program with end-primers: initial Denat. 40´´ at 98°C; Denat 12´´ at 98°C; Annealing 15´´ at 57°C; Elongation 27´´ at 72°C; final-Elongation 10´at 72°C; 32 cycles

further Tasks:

PCR-Clean-Up

analytical gelelectrophoresis of cleaned and amplifyied scFv-TEV-TMD-EYFP genecostruct

digestion of cleaned and amplifyied scFv-TEV-TMD-EYFP geneconstruct

PCR-Clean-Up of scFv-TEV-TMD-EYFP geneconstruct

Investigators: Kerstin/Stefan

Aim: cleaning of scFv-TEV-TMD-EYFP genecostruct

Materials:

PCR-Clean-Up Kit

Method:

according to manual

analytical gelelectrophoresis of cleaned and amplifyied scFv-TEV-TMD-EYFP geneconstruct

Investigators: Kerstin/Stefan

Aim: analytical gelelectrophoresis to check size of amplifyied scFv-TEV-TMD-EYFP geneconstruct in 1% agarosegel

Materials:

agarose

1xTAE-buffer

10xFD Green Buffer

Method:

1µl 10x FD Green, 1µl of amplifyied scFv-TEV-TMD-EYFP geneconstruct, water add to 10µl

1% agarosegel, 90ml

70 min at 120V

Results:

amplification worked --> DNA-bond at 1701bp

Further Tasks:

preparative digestion of pcDNA5-FRT and scFv-TEV-TMD-EYFPv geneconstruct

ligation of pcDNA5-FRT and scFv-TEV-TMD-EYFPv geneconstruct

preparative digestion of pcDNA5-FRT from

Investigators: Kerstin/Stefan

Aim: digestion of pcDNA5-FRT with NheI and ApaI to generate sticky ends for ligation with scFv-TEV-TMD-EYFPv geneconstruct

Materials:

Fast Digest NheI

Fast Digest ApaI

10x FD Green Buffer

pcDNA5-FRT(gut) from

steril water

Method:

2,5µl pcDNA5-FRT/gut (729,ng/µl) --> 1822ng

2µl NheI

2µl ApaI

3µl 10x FD Green Buffer

water add to 30µl

digestion for 2,5h at 37°C

Further Tasks:

dephosphorylation

gelelectrophoresis

gelextraction

dephosphorylation of pcDNA5FRT digested with NheI and ApaI

Investigators: Kerstin/Stefan

Aim:dephosphorylation of digestd pcDNA5-FRT to prevent religation

Materials:

Antarctic Phosphatase

10x Antarctic Phosphatase Reaction Buffer

digested pcDNA5-FRT

Method:

3,3µl 10x Antarctic Phosphatase Reaction Buffer

1µl Antarctic Phosphatase

incubation for 30min at 37°C to dephosphorylate 5´-ends of pcDNA5-FRT

heat inactivation of Phosphatase for 5min at 65°C

store at -20°C

Further Tasks:

gelelectrophoresis

gelextraction

digestion of scFv-TEV-TMD-EYFP geneconstruct

2012-08-19

ligation of digested scFv-TEV-TMD-EYFP geneconstruct and digested + dephosporylated pcDNA5-FRT

Investigators: Kerstin/Stefan

Aim: ligation of digested scFv-TEV-TMD-EYFP geneconstruct and pcDNA5-FRT

Materials:

ligation calculator: http://www.insilico.uni-duesseldorf.de/Lig_Input.html

T4 DNA ligae

ligase buffer

digested scFv-TEV-TMD-EYFP geneconstruct

digested + dephosporylated pcDNA5-FRT

Method: ligation-ratio--> 1:3

1µl T4 DNA ligase

2,5µl T4 DNA-ligase buffer

11,9µl digested + dephosporylated pcDNA5-FRT (8,4ng/µl) --> 100ng

9,67µl digested scFv-TEV-TMD-EYFP geneconstruct (10,4ng/µl) --> 100,66ng

religation-control

incubation for 1h at RT

Further Tasks:

transformation of ligation mix into XL1-blue competente E. coli cells

Transformation of ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP into XL1-blue competente E. coli cells

Investigators: Kerstin/Stefan

Materials:

Bunsen Burner, Agar Plate with Ampicilin, 37 °C heater, centrifuge,

ligase sample

icebox

competent E. coli cells (XL 1)

Method:

Transformation via manual

50µl of resuspended cell-suspension were plated on a LB-Amp-plate

incubation o.n. at 37°C

Further Tasks:

picking clones

2012-08-20

colony-pcr of ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Investigators: Kerstin/Stefan

AIM: identifying correct ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Materials:

Taq DNA-polymerase 1kb/1min

10x Taq DNA-polymerase buffer

dNTPs (10mM)

forward primer in C-terminus of CMV of pcDNA5-FRT-backbone: fp_qRT-CMV_rev (Tm=55°C)

reverse primer in C-terminus in EYFP: ps_mcherry_rev_BamHI (Tm= 59,31°C)

Thermocycler

Methods:

20µl colony-pcr mix for one clone: 2µl 10x Standard Taq-DNA-Polymerase Buffer; 0,4µl dNTPs (10mM), 0,4µl fp_qRT-CMV_rev(10µM), 0,4µl ps_mcherry_rev_BamHI (10µM); 0,1µl Taq-DNA-Polymerase; nuclease-free water add to 20µl

colony-pcr mix were adapted to 26 clones

after transferation of clone into 20µl colony-pcr mix, the rest of clone at the tip was plated on a LB-AMP-plate

LB-AMP-plate were incubated o.n. at 37°C

negative control: clon #24 from relagtion-control (ligation-ratio 1:0) from 2012-07-31

program of colony-pcr: initial Denaturation:5´at 95°C; Denaturation: 25´´ at 95°C, Annealing: 25´´ at 51°C; Elongation: 114´´ (1845bp) at 68°C; final Elongation: 5´at 86°C; 30 cycles; store at 8°C

further Taks:

gelelectrophoresis

picking positiv clones for o.n.-culture

gelelectrophoresis of colony-pcr

Investigators: Kerstin/Stefan

AIM: identifying correct ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Materials:

1 100 ml 1%agarosegel with 20 slots

1 90 ml 1% agarosegel with 9 slots

1x TAE-buffer

agarose

Method:

90 ml 1% agarosegel for 80 min at 110V

100 ml 1% agarosegel for 100 min at 110V

Results:

(positive) clones 4, 8, 12, 16 were chosen for o.n.-culture because of desired DNA-bond at appropriate 1845bp

further Tasks:

o.n.-culture of positive clones

o.n.-culture of positive ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Investigators: Kerstin/Stefan.

Materials:

4x 15 ml Falcons

LB-Medium

Ampicilin

bunsen burner

further Tasks:

miniprep of o.n.-cultures

2012-08-21

miniprep of o.n.-culture of positive ligated pcDNA5-FRT_scFv-TEV-TMD-EYFP clones

Investigators: Kerstin/Stefan

Materials:

Miniprep-Kit

Method:

according to manual

Results: concentration measurement via nanodrop

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone4= 540 ng/µl

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone8= 663 ng/µl

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone12=572ng/µl

pcDNA5-FRT_scFv-TEV-TMD-EYFP_clone16=580ng/µl

further Tasks:

sequencing of the clones 4 and 8

if they are correct ===> stable transfection

2012-08-27

Transformation of Clon4 (scFv425+TEV+TMD+YFP in pcDNA5/FRT) and Pog44 (Flp recombinase vector)

Investigator:Maria

Time: 2012-08-27 5:00pm

Materials: competent E. coli cells (XL1 blue), icebox, Bunsen burner, agar plate with ampicillin

Methods: Transformation according to manual, 2µl of Clon4 and Pog44 were used, plate incubation started 6:30pm

Results: colonies ready for picking and overnight culture

Further tasks: Picking colonies

Overnight culture of CMV-Venus

Investigator:Maria

Time: 2012-08-27 5:00pm

Materials: glycerol stock CMV-Venus, 50ml LB media with chloramphenicol

Methods: inoculation of LB-chloramphenicol

Results: overnight culture for endotoxin free DNA preparation

Further tasks: endotoxin free preparation

2012-08-28

Endotoxin free preparation of CMV-venus

Investigator:Maria

Time: 2012-08-28 11:00 am

Materials: endotoxin free Mediprep kit, overnight culture CMV-Venus

Methods: according to manual

Results: CMV-Venus with --- ng/µl

Further tasks: transient transfection of CHO cells

Overnight culture Clon4 and Pog44

Investigator:Maria

Time: 2012-08-28 5:00 am

Materials: colonies of Clon4 and Pog44, 5ml LB media with ampicillin

Methods: inoculation of LB-ampicillin

Results: overnight culture for endotoxin free DNA preparation

Further tasks: endotoxin free preparation

2012-08-27

cell culture;

Investigator:Kerstin/Stefan

Time: 2012-08-29 11:00 am

Topic: cell culture

Materials: 6-well plates;

transfection of 6-well plate with different PEI amounts

- well 1) 1 fold PEI; 2) 1,5 fold PEI; 3) 2 fold PEI; 4) 2,5 fold PEI; 5) 3 fold PEI; 6) 3,5 fold PEI of DNA amount

NOTE: due to not conclusive experiments, it was decided to keep the original protocol with 2,5 fold PEI

2012-08-29

cell culture;

Investigator:Kerstin/Stefan

Time: 2012-08-29 11:00 am

Materials: cell-culture flask (75cm^2); 6-well plates; ibidi-dishes

Topic: cell culture;

thowing CHO cells, cultured w/o zeocin

passaging (1:10) of CHO’s one flask w/ and one w/o zeocin

seeding CHO’s in 6-well 2*10^5 cells/well

seeding CHO’s in ibidi dishes 5*10^4 cells/well

transfection of clon 4 (540ng/µl) (small construct) of CHO’s in ibidi dishes, 1µg(DNA):2,5µl(PEI) = 0,25µg DNA:0,625µl PEI

transfection of 6-well plate with different DNA amounts

- well 1) 1µg(0.5µgmVenus+0,5µg AID) DNA; 2) 1,5µg DNA; 3) 2µg DNA; 4) 3µg DNA; 5) 4µg DNA; 6) 5µg DNA with 2,5 fold PEI

2012-08-31

cell culture;

Investigator:Kerstin/Stefan

Time: 2012-08-31 09:00 am

Topic: cell-culture

Methods:

passaging of thawed CHO’s w/ and w/o zeocin (of 29.08.2012 seeded cells)

imaging (longtime imaging over 48 hours) of the transfected cells (30.08.2012) CHO’s in ibidi dishes with m-Venus+wild Type AID

2012-08-30

cell culture;

Investigator:Kerstin/Stefan

Time: 2012-08-30 09:00 am

Topic: cell-culture

change of medium of thawed CHO’s w/ zeocin

transfection of seeded (29.08.2012) CHO’s in ibidi dishes with m-Venus (250ng) and wild type AID (250ng)

z-stack of clon 4(small construct)

2012-08-29

Endotoxin free preparation of Clon4 and Pog44

Investigator:Rico, Tom, Chris

Time: 2012-08-29

Materials: endotoxin free Mediprep kit, overnight culture Clon4, Pog44

Methods: according to manual

Results: Clon 4 with --- ng/µl, Pog44 with ----ng/µl

Further tasks: stable transfection of CHO cells

2012-08-30

Primer design for Biobricks with RCF 25 standard

Investigator:Maria

Time: 2012-08-30 6:30 am

Materials: Geneious

Results: Primer big construct

Primer 1.1 and 1.2: Signal peptide-Nanobody-Fc

Primer 2.1 and 2.2: TEV-LoxP-TMD-mcherry-LoxP

Primer 3.1 and 3.2: Nanobody

Primer 4.1 and 4.2(=1.2): Fc

Primer5.1 and 5.2: TMD-mcherry

small construct, mutagenese PCR

Primer 1.RCF25 and 2.mut: RCF25 prefix and 1st mutation PstI in scFV

Primer 3.mut and 4.mut: 1st and 2nd mutation PstI in scFV

Primer 5.mut and &.RCF25: 2nd mutation and RCF25 suffix

Primer scFv: Primer 7.Rcf25 prefix and 8.RCF25 suffix

Further tasks: PCR and seuqencing

Sequence alignment of Geneart construct

Time: 2012-08-30 12 pm

Materials: Geneious, sequences delivered by Geneart

Results: sequence correct, no mutations, no undesired restriction sites

Transformation of Geneart construct Nanobody-Fc-LoxP-TEV-TMD-mcherry

Investigator:Maria

Time: 2012-08-30 17 pm

Materials: competent E. coli cells (XL1 blue), icebox, Bunsen burner, agar plate with kanamycin

Method: Transformation according to manual, 2µl of Geneart construct

Results: colonies ready for picking and overnight culture

Further tasks: Picking colonies

2012-08-31

Control of Primer and Ordering Primers

Investigator:Sascha, Maria

Time: 2012-08-31 11 am

Materials: Geneious

Results: 17 primer for Biobrick assembly with RCF25 standard

Further tasks: PCR

Overnight culture Geneart construct

Investigator:Maria

Time: 2012-08-31 5:00pm

Materials: colonies Geneart construct, 5ml LB media with kanaymycin

Methods: inoculation of LB-kanaymycin

Results: overnight culture for mini prep

Further tasks: Mini prep

Virus

2012-08-13

Topic: PCR

Investigator: Xenia

Aim:test primer prr_VP2_pstI_2 (reverse primer)

Materials:

primer combination:

prr_VP2_pstI_2 (reverse primer) and prf_XbaI_koz_GGGGG_VP2 (forward primer)-> myc-

prr_VP2_pstI_2 (reverse primer) and prf_XbaI_koz_GGGGG_myc_VP2(forward primer)--> myc+

Method:

polymerase chain reaction

Mastermix

reagenz	volumen [µL]
HF buffer	5
dNTPs (NEB)	1.25
primer (prr_VP2_pstI_Temp68)	1.0
Primer (prf_XbaI_koz_GGGGG_VP2/prf_XbaI_koz_GGGGG_myc_VP2)	1.0
DNA (plasmid)	1.0
Phusion polymerase	1.0
water	33.75

program

step	temperature [°C]	duration [s]	cycles
denaturation	98	30	1
denaturation	98	10	30
annealing	70	40	30
elongation	72	40	30
final elongation	72	300	1
cooling	4	∞	1

Further tasks:

agarose gel electrophoresis

2012-08-14

Topic: PCR von VP2

Investigators: Tobias

Materials:

Phusion polymerase

Templat: Plasmid (P10_pSB1C3_001_CMV_DARPin_ML_VP2/3_587KO_6xHis)

primer:

- prr_VP2_pstI_2 (reversed) and prf_XbaI_koz_GGGGG_VP2 (FP1)-> myc-

- prr_VP2_pstI_2 (reversed) and prf_XbaI_koz_GGGGG_myc_VP2(FP2)--> myc+

Method:

polymerase chain reaction

Mastermix

reagenz	volumen [µL]
HF buffer	10
dNTPs (NEB)	1
primer (prr_VP2_pstI_2)	2.0
Primer (prf_XbaI_koz_GGGGG_VP2/prf_XbaI_koz_GGGGG_myc_VP2)	2.0
DNA (plasmid)	1.0
Phusion polymerase	0.5
water	33.5

Program

step	temperature [°C]	duration [s]	cycles
denaturation	98	30	1
denaturation	98	10	30
annealing	65	40	30
elongation	72	40	30
final elongation	72	300	1
cooling	4	∞	1

Further tasks:

PCR-clean up

2012-08-15

Topic: PCR-clean up

Investigator: Tobias

Materials: NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)

Methods:

CleanUp-protocol

Results:

-myc-product: 18 ng/µL --> 14 nM

+myc-product: 7 ng/µL --> 5 nM

Further tasks:

digestion

Topic: ligation, transformation

Investigator: Tobias

Materials and Methods:

the ligation and the transformation were done using the standard protocol

Further tasks:

picking clones

2012-08-17

Topic: pick clones, DNA preparation, gel electrophoresis

Investigators: Kathi

Materials:

LB-medium

GeneJET Plasmid Miniprep Kit

gel electrophoresis

Methods:

Pick clones, eight hours incubation (LB-medium, 37 °C)

perform the DNA preparation

control (agarose gel electrophorese)

results:

p10_PsB1c3_CMV_kozak_sortase: c= 185 ng/µL v= 50 µL

p10_PsB1c3_CMV_kozak_sortase_myc: c= 202 ng/µL v= 50 µL

further tasks: test digestion

2012-08-28

purification of plasmid DNA

Investigators: Xenia and Kathi

Materials methods

endotoxin free preparation - Plasmid Plus Midi Kit (QIAGEN)- elution volume 200 µL

Results:

concentration of plasmids

P01 (mVENUS linked on VP1): 1776.7 ng/µL

P05 (VP2 knock out) : 2336.2 ng/µL

P06 (VP1 knock out): 1256.7 ng/µL

phelper: 1628 ng/µL

psB1c (GOU=CFP): 493 ng/µL

Further tasks:

test digestion

gel electrophoresis

test digestion, Gelelectrophoresis

Investigator : Xenia

Materials and Method:

test digestion

15 µL water + 2 µL 10x FD green buffer + 2 µL DNA (300 ng/µL) + 1 µL of each enzyme

- psB1c (GOI=CFP): SpeI, XbaI und ApaI

- phelper: NheI, SpeI, ApaI

- P06 (VP1 knock out): XbaI, ApaI

- P05 (VP2 knock out): XbaI, ApaI

- P01 (YFP linked on VP1): XbaI, ApaI

1 hour at 37 °C

gel electrophoresis

Results:

fragments calculated in Genious:

psB1c (GOI=CFP): 4069 bp, 284 bp, 219 bp

phelper: 6242 bp, 4011 bp, 906 bp, 492 bp

P06 (VP1 knock out): 2119 bp, 290 bp, 631 bp, 2630 bp, 821 bp

P05(VP2 knock out): : 2119 bp, 290 bp, 631 bp, 2630 bp, 821 bp

P01(YFP linked on VP1): 2566 bp, 2169 bp, 821 bp, 323 bp

fragments - gel electrophoresis

phelper: 6242 bp, 4011 bp, 906 bp, 492 bp

P06 (VP1 knock out): 2119 bp, 290 bp, 631 bp, 2630 bp, 821 bp + ~ 3000 bp

P05(VP2 knock out): : 2119 bp, 290 bp, 631 bp, 2630 bp, 821 bp + ~ 3000 bp

P01(YFP linked on VP1): 2566 bp, 2169 bp, 821 bp, 323 bp

psb1c3 (GOI=CFF): other fragments in the gel electrophoresis than calculated in Genious

Further Tasks:

sequences of the psb1c3 (GOI=CFP)-plasmid

2012-08-29

Topic: transfection of plasmids

Investigators: Kerstin and Stefan

Aim:

The plasmids were transfected in HEK-cells with the following plasmids:

- pSB1C: GOI is CFP

- phelper

- P01: YFP linked on VP1

- p06: VP1 knock out

Materials:

Medium: opti Mem

transfection reagenz: PEI

15 µg DNA per each transfection

Methods:

transfection preparation

- 1875 ng per plasmid; add 200 µL opti MEM-medium per each transfection

- 4.8 * DNA --> 72 µL PEI add 200 µL per each transfection

three times 10 s mixing

2 min RT

centrifagation

give the PEI preparation to the DNA preparation

mixing

15 min RT

give the transfection preparation to the HEK AAV 293 cells

Further tasks

change medium (DMEM) after 16 hours

after three days virus maturation, preparation of the primary virus stocks

2012-08-30

Topic: change medium

Investigators: Kerstin and Stefan

Aim: change the medium of the transfected cells

Materials:

PBS

DMEM

Methods:

aspirate the old medium

washed with PBS

give new DMEM medium to the cells

Further tasks

after three days virus maturation - virus preparation on 02.09.2012

@@ Line 6,187: / Line 6,187: @@
 * looking for primer for GATC-sequencing of positive pcDNA5-FRT_scFv-TEV-TMD-EYFP_clones
+===<p style="background-color: rgb(240, 20, 70);">2012-08-10</p>===
+<p style="background-color: rgb(238, 221, 130); font-weight:bold;"> preparing ligated pcdna5frt-scfv-tev-tmd-eyfp-construct for ordering sequencing by GATC</p>
+<b>Investigators:</b> Sascha<br>
+<b>Materials:</b>
+*dilute DNA (clons 3,8,21) according to GATC requirements
+*Geneious<br>
+*Using GATC standard-primer pcdna3.1FP.1 and pcdna3.1RP.1<br>
+<b>Further Tasks:</b><br>
+* analysis of sequencing results
 ===<p style="background-color: rgb(240, 20, 70);">2012-08-13</p>===

Team:Potsdam Bioware/Lab/Labjournal/August

From 2012.igem.org

Revision as of 22:33, 26 September 2012

Contents

AID

2012-08-01

2012-08-02

2012-08-03

2012-08-04

2012-08-06

2012-08-07

2012-08-08

2012-08-09

2012-08-10

2012-08-11

2012-08-13

2012-08-14

2012-08-15

2012-08-16

2012-08-17

2012-08-20

2012-08-21

2012-08-22

2012-08-23

2012-08-24

2012-08-27

2012-08-28

2012-08-29

2012-08-30

2012-08-31

Antibody

2012-08-02

2012-08-03

2012-08-05

2012-08-06

2012-08-07

2012-08-08

2012-08-10

2012-08-13

2012-08-15

2012-08-15

2012-08-17

2012-08-18

2012-08-19

2012-08-20

2012-08-21

2012-08-27

2012-08-28

2012-08-27

2012-08-29

2012-08-31

2012-08-30

2012-08-29

2012-08-30

2012-08-31

Virus

2012-08-13

2012-08-14

2012-08-15

2012-08-17

2012-08-28

2012-08-29

2012-08-30