From 2013.igem.org

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Revision as of 17:52, 1 October 2013

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	ddH2O

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
4 µl 2-log DNA Ladder

Expectations

Lane 2: 479 + 2,581 + 6,000 bp
Lane 3: 1,874 + 6,000 bp
Lane 4: 2,160 + 3,650 + 6,000 bp
Lane 5: 1,823 + 6,000 bp
Lane 6: 1,199 + 2,682 + 6,000 bp
Lane 7: 898 + 6,000 bp
Lane 8: 693 + 6,000 bp

We received all expected fragments. Assembly of biobricks was 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 ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

Worked as expected.

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	ddH2O

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
4 µl 2-log DNA ladder

Expectations (upper gel)

Lane 2-4: 1,008 bp
Lane 5-7: 927 bp

Expectations (lower gel)

Lane 2-3: 903 bp
Lane 4-7: 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 was believed 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 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 and incubated ON 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 ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

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 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 RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

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.
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 ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

Two samples of each plasmid preparation showed the expected fragments.

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.

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 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 RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

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+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 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 ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

Most of the preparations resulted in the expected fragments, all others were discarded.

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 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.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).

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. Additionally the remaining ligation preparation was transformed in E. coli DH5α?????.

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 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.
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

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 RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

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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Team:Marburg/Notebook:July

From 2013.igem.org

Revision as of 17:52, 1 October 2013

Notebook: July