From 2013.igem.org

Notebook: July

01.07.2013

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	ddbidest. H2O

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.

02.07.2013

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 EcoRI and PstI

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 ddbidest. H2O

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.

15.07.2013

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.

16.07.2013

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	ddbidest. H2O

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
BBa_K1071008	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 is thought to the accurate.

17.07.2013

Digest

Investigator: Alex

Aim: Digest of pSB1A3-iBB10/11/12/13+9 with XbaI and PstI, and pSB1C3-iBB4 with SpeI and PstI.

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 ddbidest. H2O

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 on ice, 90 sec 42 °C, 45 min 37 °C + 900 µl LB) and incubated ON at 37 °C.

18.07.2013

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.

19.07.2013

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)).

22.07.2013

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 bidest. H2O

Sample:

Digestion of 5 µl iBB4 with SpeI and PstI.

Digestion of 2 µl iBB10+9 and 4 µl iBB13+9 with XbaI and PstI.

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 bidest. 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 on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB_CM, overnight at 37 °C.

23.07.2013

Digest

Investigator: Dominik

Aim: Test digest of the plasmids pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9 with EcoRI and PstI

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 ddbidest. H2O

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.

24.07.2013

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 ddbidest. H2O

1000 ng Plasmid (pSB1C3-iBB6315)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddbidest. H2O

1000 ng Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddbidest. H2O

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).

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 ddbidest. H2O

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.
The plates were incubated over night at 37 °C.

25.07.2013

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 EcoRI and PstI

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 ddbidest. H2O

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 at 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.

26.07.2013

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 ddbidest. H2O

1000 ng Plasmid (pSB1C3-iBB6315)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddbidest. H2O

1000 ng Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddbidest. H2O

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).

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 ddbidest. H2O

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 EcoRI and PstI

1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddbidest. H2O

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.

30.07.2013

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 ddbidest. H2O

1000 ng Plasmid (pSB1C3-iBB6315)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
ad 20 µl ddbidest. H2O

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.

31.07.2013

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 ddbidest. H2O

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.
The plates were incubated over night at 37 °C.

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 EcoRI and PstI