PCR
Investigator:
Alex
Aim:
Check correct assembly of the plasmid for antibody production
Volume
Reagent
Temp (°C)
Time
1 µl
pSB1A3-iBB486476315
95
3 min
0.5 µl
Primer forward
95
30 sec
0.5 µl
Primer reverse
60
30 sec
x30
0.5 µl
dNTPs
72
42 sec
0.5 µl
Phusion polymerase
72
5 min
5 µl
Phusion buffer 5x
4
Hold
17 µl
ddH2O
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Lane
Content
Expectations
2
PCR with iMR08 and iMR37
479 bp
3
PCR with iMR34 and iMR37
1874 bp
4
PCR with iMR12 and iMR37
2160 bp + 3650 bp
5
PCR with iMR31 and iMR38
1823 bp
6
PCR with iMR11 and iMR38
1199 bp + 2682 bp
7
PCR with iMR5 and iMR38
898 bp
8
PCR with iMR9 and iMR38
693 bp
We receive all expected fragments.
Assembly of biobricks is successful.
Miniprep
Investigator:
Dominik
Aim:
preparation of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.
The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.
Digest
Investigator:
Dominik
Aim:
Test digest of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 with Eco RI and Pst I
1 µl Plasmid (pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddH2O
The samples were incubated for 1 h at 37° C.
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Lane
Content
Expectations
2
pSB1A3-iBB10+9
5300 bp
3
pSB1A3-iBB11+9
3300 bp
4
pSB1A3-iBB12+9
1300 bp
Worked as expected.
3 out of 6 samples positive.
Sequencing
Investigator:
Franzi
Aim:
Construction of antibody-vector → Sequencing.
Sequencing pSB1A3 iBB486476315 K1 with primer 33 (iBB8 fw) AGBOOOK 275 and primer 34 (iBB8 rv) AGBOOOK 276.
PCR
Investigator:
Alex
Aim:
Check ligation of iBB10/11/12/13 + iBB9 in pSB1A3
Volume
Reagent
Temp (°C)
Time
1 µl
Template DNA
95
3 min
0.5 µl
Primer forward
95
30 sec
0.5 µl
Primer reverse
60
30 sec
x60
0.5 µl
dNTPs
72
1 min
0.5 µl
Phusion polymerase
72
5 min
5 µl
Phusion buffer 5x
4
Hold
17 µl
ddH2O
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Upper gel
Lane
Content
Expectations
2-4
pSB1A3-iBB10+9
1008 bp
5-7
pSB1A3-iBB11+9
927 bp
Lower gel
Lane
Content
Expectations
2-3
pSB1A3-iBB12+9
903 bp
4-7
pSB1A3-iBB13+9
888 bp
The upper gel showed the expected bands, whereas these bands were missing in the lower gel.
Sequencing
Investigator:
Alex
Aim:
Evaluate sequencing result of iBB8 in pSB1A3-iBB486476315
DNA / Biobrick
Used primer
Identities
Gaps
BBa_K1071008
AB_plasmid_8 forward
941/941 (100%)
0
AB_plasmid_8 reverse
917/918 (100%)
1
Due to unreliabilities of the chain stop method by Sanger at the end of a DNA sequence, the detected gap can be considered as uncertain. This thesis is supported by 100% identity in the complementary strand.
The sequenced biobrick was believed to the accurate.
Digest
Investigator:
Alex
Aim:
Digest of pSB1A3-iBB10/11/12/13+9 with Xba I and Pst I, and pSB1C3-iBB4 with Spe I and Pst I.
2 µl Plasmide DNA
0.6 µl Enzyme 1 (XbaI or SpeI, resp.)
0.6 µl Enzyme 2 (PstI)
2 µl CutSmart
14.8 µl ddH2O
Ligation and transformation
Investigator:
Alex
Aim:
Assemble iBB4 in vectors pSB1A3-iBB10/11/12/13
Insert (iBB4) und plasmids were transformed using a 3:1 ratio.
Ligation samples were incubated ON at 16 °C.
Ligation products (pSB1A3-iBB4+10/11/12/13+9) were transformed into chemical competent DH5α cells (30 min ice, 90 sec 42°C, 45 min 37°C + 900 µl LB) and incubated ON at 37 °C.
Inoculation
Investigator:
Alex
Aim:
Inoculation of colonies for midi plasmid preparation
Several clones of pSB1A3-iBB486476315/-iBB496315/-iBB395416 were picked and inoculated in 100 ml LB medium containing ampicillin.
Cultures were incubated ON at 37 °C.
Midiprep
Investigator:
Christian
Aim:
Preparing the vectors for Phaeo → Midiprep of antibody-vector, PNR-test-vector and PfcpB-test-vector.
Midiprep of pSB1A3 iBB486476315, pSB1A3 iBB496315 and pSB1A3 iBB395416 (“QIAprep Spin Midiprep Kit” (Qiagen, Düsseldorf)).
Digest
Investigator:
Christian
Aim:
Construction of signal peptide-vectors → Digestion of pSB1C3 iBB4, iBB10+9 and iBB13+9.
X µl DNA template
1 µl enzyme 1
1 µl enzyme 2
2 µl Cut Smart buffer
ad 20 µl H2O
Sample:
Digestion of 5 µl iBB4 with Spe I and Pst I.
Digestion of 2 µl iBB10+9 and 4 µl iBB13+9 with Xba I and Pst I.
The samples were incubated for 1 h at 37° C.
Ligation
Investigator:
Christian
Aim:
Construction of signal peptide-vectors → Ligation of iBB10+9 and iBB13+9 into pSB1C3 iBB4.
1 µl vector (pSB1C3 iBB4)
either 5 µl iBB10+9
or 5 µl iBB13+9
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
11 µl H2O
The samples were incubated for 2 h at RT.
Transformation
Investigator:
Franzi
Aim:
Construction of signal peptide-vectors → Transformation of pSB1C3 iBB4-10-9 and pSB1C3 iBB4-13-9 into E. coli .
Transformation of the ligations into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LBCM , oN 37°C.
Digest
Investigator:
Dominik
Aim:
Test digest of the plasmids pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9 with Eco RI and Pst I
1 µl Plasmid (pSB1C3-iBB4+iBB11+iBB9 and pSB1C3-iBB4+iBB12+iBB9)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddH2O
The samples were incubated for 1 h at 37° C.
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Lane
Content
Expectations
2-5
pSB1C3-iBB4+11+9
1349 bp
6-8
pSB1C3-iBB4+12+9
1325 bp
Worked as expected.
Inoculation
Investigator:
Dominik
Aim:
inoculation of colonies for plasmid preparation
4 colonies of pSB1C3-iBB4+iBB10+iBB9 and 4 colonies of pSB1C3-iBB4+iBB13+iBB9 were inoculated in 5 ml LB medium containing chloramphenicol.
Digest
Investigator:
Dominik
Aim:
Digest of pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9, pSB1C3-iBB6315 and pSB1A3.
1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)
0.5 µl EcoRI
0.5 µl SpeI
2 µl CutSmart
ad 20 µl ddH2O
1000 ng Plasmid (pSB1C3-iBB6315)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddH2O
1000 ng Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddH2O
The samples were incubated for 1 h at 37° C.
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Lane
Content
Expectations
2
pSB1C3-iBB4+11+9
1349 bp
3
pSB1C3-iBB4+12+9
1325 bp
4
pSB1C3-iBB6315
1082 bp
5
pSB1A3
2000 bp
All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Ligation
Investigator:
Dominik
Aim:
Assemble the biobricks iBB4+iBB11+iBB96315 and iBB4+iBB12+iBB96315 into the vector pSB1A3.
2 µl vector DNA (pSB1A3)
5 µl insert 1 DNA (iBB6315)
5 µl insert 2 DNA (iBB4+iBB11+iBB9, iBB4+iBB12+iBB9)
2 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase
4 µl ddH2O
The samples were incubated for 2h at 16 °C.
Transformation
Investigator:
Dominik
Aim:
Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.
The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.
Sequencing
Investigator:
Dominik
Aim:
Complete sequencing of pSB1A3-iBB496315.
8 new sequencing samples were sent out.
Miniprep
Investigator:
Dominik
Aim:
preparation of the plasmids pSB1A3-iBB4+iBB10+iBB9 and pSB1A3-iBB4+iBB13+iBB9.
The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.
Digest
Investigator:
Dominik
Aim:
Test digest of pSB1C3-iBB4+iBB10+iBB9 and pSB1C3-iBB4+iBB13+iBB9 with Eco RI and Pst I
1 µl Plasmid (pSB1C3-iBB4+iBB10+iBB9 and pSB1C3-iBB4+iBB13+iBB9)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddH2O
The samples were incubated for 1 h at 37° C.
Inoculation
Investigator:
Dominik
Aim:
inoculation of colonies for plasmid preparation
4 colonies of pSB1A3-iBB4+iBB11+iBB96315 and 4 colonies of pSB1A3-iBB4+iBB12+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol.
Competent cells
Investigator:
Dominik
Aim:
Overnight culture for making new aliquots of competent cells.
For making new aliquots of E. coli DH5α cells 50 ml LB medium were inoculated with 500 µl of an E. coli DH5α culture.
Digest
Investigator:
Dominik
Aim:
Digest of pSB1C3-iBB4+iBB10+iBB9, pSB1C3-iBB4+iBB13+iBB9, pSB1C3-iBB6315 and pSB1A3.
1000 ng Plasmid (pSB1C3-iBB4+iBB10+iBB9, pSB1C3-iBB4+iBB13+iBB9)
0.5 µl EcoRI
0.5 µl SpeI
2 µl CutSmart
ad 20 µl ddH2O
1000 ng Plasmid (pSB1C3-iBB6315)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddH2O
1000 ng Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddH2O
The samples were incubated for 1 h at 37° C.
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Lane
Content
Expectations
2
pSB1C3-iBB4+10+9
1430 bp
3
pSB1C3-iBB4+13+9
1310 bp
4
pSB1C3-iBB6315
1082 bp
5
pSB1A3
2000 bp
Two expected fragments are present. The relevant fragments are cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Ligation
Investigator:
Dominik
Aim:
Assemble the biobricks iBB4+iBB10+iBB9, iBB4+iBB13+iBB9 and iBB96315 into the vector pSB1A3.
2 µl vector DNA (pSB1A3)
5 µl insert 1 DNA (iBB96315)
5 µl insert 2 DNA (iBB4+iBB10+iBB9,iBB4+iBB13+iBB9)
2 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase
4 µl ddH2O
The samples were incubated for 1h at 16 °C.
Resulting in the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.
Miniprep
Investigator:
Dominik
Aim:
preparation of the pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.
The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.
Digest
Investigator:
Dominik
Aim:
Test digest of ppSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 with Eco RI and Pst I
1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddH2O
The samples were incubated for 1 h at 37° C.
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
Lane
Content
Expectations
2
pSB1A3-iBB4+10+96315
2392 bp
3
pSB1A3-iBB4+12+96315
2287 bp
4
pSB1C3-iBB39
913 bp
5
pSB1A3-iBB49
1142 bp
An expected double band is shown in Sample 1 (Vector + Insert). The other sample do not exhibit the expected band and are therefore rejected.
Competent cells
Investigator:
Dominik
Aim:
New aliquots of competent cells (E. coli DH5α).
About 80 new aliquots were made.
Digest
Investigator:
Dominik
Aim:
Digest of pSB1A3-iBB49 and pSB1C3-iBB6315.
1000 ng Plasmid (pSB1A3-iBB49)
0.5 µl PstI
0.5 µl SpeI
2 µl CutSmart
ad 20 µl ddH2O
1000 ng Plasmid (pSB1C3-iBB6315)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddH2O
The samples were incubated for 1 h at 37° C.
Inoculation
Investigator:
Dominik
Aim:
inoculation of colonies for plasmid preparation
4 colonies of pSB1A3-iBB4+iBB10+iBB96315 and the only single colony of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol.
Ligation
Investigator:
Dominik
Aim:
Ligation of pSB1A3-iBB49 and iBB6315.
2 µl vector DNA (pSB1A3-iBB49) (40 ng)
5 µl insert DNA (iBB6315) (150 ng)
2 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase
9 µl ddH2O
The samples were incubated for 1h at 16 °C.
Transformation
Investigator:
Dominik
Aim:
Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB49+iBB6315.
The chemo competent E. coli DH5α cells were transformed with the plasmid pSB1A3-iBB49+iBB6315 and plated on LB-Amp-plates.
Miniprep
Investigator:
Dominik
Aim:
Preparation of the pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.
The culture of pSB1A3-iBB4+iBB13+iBB96315 had a pinkish color and therefore was discarded. The plasmid pSB1A3-iBB4+iBB10+iBB96315 was isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.
Digest
Investigator:
Dominik
Aim:
Test digest of pSB1A3-iBB4+iBB10+iBB96315 with Eco RI and Pst I
1 µl Plasmid (pSB1A3-iBB4+iBB10+iBB96315)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddH2O
The samples were incubated for 1 h at 37° C.
Gel electrophoresis
Gel substances
1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
x µl Hyper Ladder
Expectations
Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp
Worked as expected.
Sequencing
Investigator:
Dominik
Aim:
Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.
8 samples were sent out for sequencing.
Inoculation
Investigator:
Dominik
Aim:
inoculation of colonies for plasmid preparation
Both colonies of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing ampicillin.
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