From 2013.igem.org

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Revision as of 13:48, 3 October 2013

Notebook: May

02.05.2013

Investigator: Franzi

Aim: Construction of BioBricks → Inoculation of pSB1C3 iBB8 for miniprep.

5 colonies of pSB1C3 iBB8 inoculated in 5 ml LB_cm each, 2 colonies of 2xNR HC + LC inoculated in 5 ml LB_amp each. Incubation overnight at 37° C.

03.05.2013

Miniprep

Investigator: Franzi

Aim: Construction of BioBricks → Miniprep of pSB1C3 iBB8.

5 samples of pSB1C3 iBB8 and 2 samples of 2xNR HC + LC were isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)).

PCR

Investigator: Franzi

Aim: Preparation of BioBrick-templates → Test-PCR of 2xNR HC+LC.

Volume	Reagent	Temp (°C)	Time
0,5 µl	Phusion-Polymerase	95	3 min
0,5 µl	Primer Cl4mA_l fw	95	30 sec
0,5 µl	Primer Cl4mA_l rv	65	30 sec	x30
1 µl	Template 2xNR HC + LC (9,2 ng/µl)	72	45 sec
0,5 µl	dNTPs	72	7 min
5 µl	5xGC buffer	4	Hold
17 µl	ddH20

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

All positive.

Digest

Investigator: Franzi

Aim: Construction of BioBricks → Control digestion of pSB1C3 iBB8.

1 µl DNA template
0,5 µl EcoRI
0,5 µl PstI
1 µl buffer O

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

iBB8 K1 + 2 chosen for further work.

06.05.2013

Transformation

Investigator: Franzi

Aim: Preparation of pSB1A3 → Transformation of pSB1A3 J04450.

3 µl of pSB1A3 J04450 (2012) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 60 min 37°C with 900 µl LB), concentrated and plated on LB_amp, overnight 37°C.

07.05.2013

Inoculation

Investigator: Franzi

Aim: Preparation of pSB1A3 → Inoculation of pSB1A3 J04450 for miniprep.

Two red colonies were picked and inoculated in 5 ml LB_amp each. Incubation overnight at 37° C.

Sequencing

Investigator: Franzi

Aim: Construction of iBB2 (cat) → Examination of sequencing results.

1C3 iBB1 K1 + K2 correct

iBB2 K2 + 4: both positive

08.05.2013

Miniprep

Investigator: Franzi

Aim: Preparation of pSB1A3 → Miniprep of pSB1A3 J04450

Miniprep of the cultures inoculated the previous day (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.

Digest

Investigator: Franzi

Aim: Preparation of pSB1A3 → Digestion of pSB1A3 J04450 with EcoRI and PstI.

1 µl DNA template
1 µl EcoRI-HF
1 µl PstI-HF
3,6 µl Cut Smart buffer

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.

13.05.2013

Digest

Investigator: Franzi

Aim: Construction of antibody-vector → Digestion of various BioBricks.

5 µl DNA template
0,5 µl enzyme 1
0,5 µl enzyme 2
2 µl Cut Smart buffer
12 µl H2O

Samples:

IBB4 K1, iBB6 K1, iBB7 K3 and iBB3 K1 were digested with EcoRI-HF and SpeI-HF.

iBB8K1, iBB4K1, iBB6K1 and iBB1K1 were digested with XbaI and PstI-HF.

The samples were incubated for 1 h at 37° C. Then 1 µl CIP was addend and the samples were incubated for another 1 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of antibody-vector → Combination of two BioBricks into vector pSB1A3.

40 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
120 ng insert DNA (variable)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

Samples:

iBB4 EcoRI/SpeI with iBB8 XbaI/PstI

iBB6 EcoRI/SpeI with iBB4 XbaI/PstI

iBB7 EcoRI/SpeI with XbaI/PstI

iBB3 EcoRI/SpeI with XbaI/PstI

The samples were incubated for 1 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of Antibody-vector → Transformation of combined 2 BioBricks in pSB1A3 into E. coli DH5α.

Complete ligation samples (see above) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 20 min 37°C with 900 µl LB), concentrated and plated on LB_amp, overnight 37°C.

14.05.2013

Inoculation

Investigator: Franzi

Aim: Construction of antibody-vector → Inoculation of transformants.

Colonies from iBB4+8, iBB6+4 and iBB3+1 were picked and inoculated in 5 ml LB_amp, respectively.

Ligation

Investigator: Franzi

Aim: Construction of antibody-vector → Ligation of iBB7 and iBB6 into pSB1A3.

Ligation of iBB7 and iBB6 was repeated.

40 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
120 ng insert DNA
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 1 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of Antibody-vector → Transformation of combined 2 BioBricks in pSB1A3 into E. coli DH5α.

Complete ligation samples (see above) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 20 min 37°C with 900 µl LB), concentrated and plated on LB_amp, overnight 37°C.

15.05.2013

Digest

Investigator: Franzi

Aim: Construction of antibody-vector → Repeat of digestion of various BioBricks (13.05.13).

Liquid cultures and transformation samples from previous day were discarded because of red colonies (Insert J04450). Digestion of BioBricks repeated like 13.5.13, but without CIP.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of antibody-vector → Repeat of combination of two BioBricks into vector pSB1A3.

Ligation like 13.5.13.

Transformation

Investigator: Franzi

Aim: Construction of Antibody-vector → Repeat of transformation of combined 2 BioBricks in pSB1A3 into E. coli DH5α.

Transformation like 13.5.13.

Inoculation

Investigator: Franzi

Aim: Preparation of pSB1A3 → Inoculation of pSB1A3 J04450.

E. coli pSB1A3 J04450 was inoculated in 5 ml LB_amp, overnight 37°C.

16.05.2013

Miniprep

Investigator: Franzi

Aim: Preparation of pSB1A3 → Miniprep of pSB1A3 J04450.

PSB1A3 J04450 (see 15.05.13) was isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)).

Inoculation

Investigator: Franzi

Aim: Construction of antibody-vector → Inoculation of transformants.

4 colonies of each transformation inoculated in 5 ml LB_amp, respectively. overnight 37°C.

17.05.2013

Miniprep

Investigator: Franzi

Aim: Construction of antibody-vector → Miniprep of combined 2 BioBricks into pSB1A3.

Miniprep of liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 30 µl H2O.

Digest

Investigator: Franzi

Aim: Construction of antibody-vector → Control digestion of combined 2 BioBricks.

1 µl DNA template
1 µl EcoRI
1 µl PstI
2 µl buffer O
0,15 µl H2O

Samples:

iBB4 + iBB8

iBB6 + iBB4

iBB7 + iBB6

iBB3 + iBB1

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

Expectations

6+4: 700 bp
7+6: 1000 bp
3+1: 700 bp
4+8: 1900 bp

Digest

Investigator: Franzi

Aim: Construction of antibody-vector → Digestion of combined 2 BioBricks.

10 µl DNA template
0,5 µl enzyme 1
0,5 µl enzyme 2
2 µl Cut Smart buffer
7 µl H2O

Samples:

Digest of pSB1A3 iBB6+4 K1+2 and pSB1A3 iBB3+1 K1+4 with XbaI and PstI-HF

PSB1A3 iBB4+8 K1+4 and pSB1A3 iBB7+6 K2+3 with EcoRI-HF and SpeI-HF.

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

21.05.2013

Ligation

Investigator: Franzi

Aim: Contruction of antibody-vector → ligation of two combined 2 BioBricks into pSB1C3.

30 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
100 ng insert 1
100 ng insert 2
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

Samples:

iBB4+8 K4 + iBB6+4 K2

iBB7+6 K2 + iBB3+1 K1

The samples were incubated for 1 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of Antibody-vector → Repeat of transformation of combined 4 BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples (see above) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 60 min 37°C with 900 µl LB), concentrated and plated on LB_cm, overnight 37°C.

Transformation

Investigator: Franzi

Aim: Construction of iBB4 → Retransformation into E. coli DH5α.

Retransformation of pSB1C3 iBB4 K1 as described above.

22.05.2013

Ligation

Investigator: Franzi

Aim: Construction of antibody-vector → Repeat of ligation of two combined 2 BioBricks into pSB1C3.

Ligation of pSB1C3 iBB 4+8+6+4 repeated like 21.5.13.

Inoculation

Investigator: Franzi

Aim: Construction of iBB4 → Inoculation of transformants.

2 colonies of pSB1C3 iBB4 K1 were inoculated in 5 ml LB_cm, overnight 37°C.

23.05.2013

Miniprep

Investigator: Franzi

Aim: Construction of iBB4 → Miniprep.

Miniprep of pSB1C3 iBB4 K1 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 20 µl H2O.

Inoculation

Investigator: Franzi

Aim: Construction of iBB4 → Inoculation of transformants.

4 colonies of pSB1C3 iBB7631 inoculated in 5 ml LB_cm, oN 37°C.

Transformation

Investigator: Franzi

Aim: Construction of iBB4 → Transformation of pSB1C3 iBB4864 into E. coli DH5α.

Ligation of pSB1C3 iBB4864 transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_cm, oN 37°C.

24.05.2013

Miniprep

Investigator: Franzi

Aim: Construction of antibody-vector → Miniprep of pSB1C3 iBB7631.

Miniprep of pSB1C3 iBB7631 K1-4 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 30 µl H2O.

Digest

Investigator: Franzi

Aim: Construction of antibody-vector → Control digestion of pSB1C3 iBB7631.

1 µl DNA template
0,5 µl EcoRI-HF
0,5 µl PstI-HF
1 µl Cut Smart buffer
7 µl H2O

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

Expectations

Vector: 2150 bp
insert (iBB7631): 1610 bp

Inoculation

Investigator: Franzi

Aim: Construction of antibody-vector → Inoculation of transformants.

4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LB_cm, oN 37°C.

27.05.2013

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector and P_fcpB-test-vector → digestion of single BioBricks.

5 µl DNA template
0,5 µl enzyme 1
0,5 µl enzyme 2
2 µl Cut Smart buffer
12 µl H2O

Samples:

Digestion of pSB1C3 iBB6, iBB3, iBB5 and iBB4 with XbaI and PstI-HF

Digestion of pSB1C3 iBB6 and iBB1 with EcoRI-HF and SpeI-HF.

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

Ligation

Investigator: Franzi

Aim: Construction of P_NR-test-vector and P_fcpB-test-vector → Combination of two BioBricks into pSB1A3.

40 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
120 ng insert 1
120 ng insert 2
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

Samples:

iBB5 E/S + iBB4 X/P

iBB1 E/S + iBB6 X/P

iBB6 E/S + iBB3 X/P

iBB1 E/S + iBB5 X/P

The samples were incubated for 2,5 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of P_NR-test-vector and P_fcpB-test-vector → transformation of combined 2 BioBricks into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 20 min 37°C + 900 µl LB), plated on LB_amp, oN 37°C.

Ligation

Investigator: Franzi

Aim: Construction of antibody-vector → Repeat of ligation of two combined 2 BioBricks into pSB1C3.

30 ng vector DNA
40 ng iBB48
40 ng iBB64
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl H2O

The samples were incubated for oN at 16° C

Digest

Investigator: Franzi

Aim: Preparation of pSB1C3 → digestion with EcoRI and PstI.

10 µl DNA template (pSB1C3 J04450)
0,5 µl EcoRI-HF
0,5 µl PstI-HF
2 µl Cut Smart buffer
6 µl H2O

The samples were incubated oN at 37° C.

Gel electrophoresis (done 28.05.2013)

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.

28.05.2013

PCR

Investigator: Franzi

Aim: Construction of BioBricks → Adding of pre- and suffix via PCR.

PCR of iBB9 (eGFP) with P40+41, iBB10 (SPTP1) with P42+43, iBB11 (SPTP2) with P44+45, iBB12 (SP1) with P46+47 and iBB13 (SP2) with P48+49.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion-Polymerase	95	3 min
1 µl	Primer P1 (1pmol/µl)	95	30 sec
1 µl	Primer P2 (1pmol/µl)	55	30 sec	x33
1 µl	Template	72	45 sec
1 µl	dNTPs	72	10 min
10 µl	5xGC buffer	4	Hold
35 µl	ddH20

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

Expectations

eGFP: 780 bp
SPTP1: 228 bp
SPTP2: 147 bp
SP1: 123 bp
SP2: 108 bp

PCR was repeated (too much template), template diluted 1:50.

Transformation

Investigator: Franzi

Aim: Construction of antibody-vector → Transformation of pSB1C3 iBB4864 into E. coli DH5α.

Ligation of pSB1C3 iBB4864 was transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

Inoculation

Investigator: Franzi

Aim: Construction of P_NR-test-vector and P_fcpB-test-vector → Inoculation of transformants for miniprep.

2 colonies of each transformation (27.05.13) were inoculated in 5 ml LB_amp, oN 37°C

29.05.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_NR-test-vector and P_fcpB-test-vector → Miniprep of combined 2 BioBricks in pSB1A3.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 30 µl H2O.

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector and P_fcpB-test-vector → Control digestion of combined 2 BioBricks in pSB1A3.

1 µl DNA templates (see above)
0,5 µl EcoRI
0,5 µl PstI-HF
1 µl Cut Smart buffer
7 µl H2O

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

Expectations

6+3: 500 bp
1+5: 700 bp
5+4: 750 bp
1+6: 650 bp

PCR

Investigator: Franzi

Aim: Construction of BioBricks → Cleaning of PCR-products.

Gel of repeated PCR of iBB9 (eGFP) with P40+41, iBB10 (SPTP1) with P42+43, iBB11 (SPTP2) with P44+45, iBB12 (SP1) with P46+47 and iBB13 (SP2) with P48+49.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

Expectations

eGFP: 780 bp
SPTP1: 228 bp
SPTP2: 147 bp
SP1: 123 bp
SP2: 108 bp

All positive.

Digest

Investigator: Franzi

Aim: Construction of BioBricks → Digestion with EcoRI and PstI.

29,5 µl DNA templates (PCR-products)
1 µl EcoRI-HF
1 µl PstI-HF
3,5 µl Cut Smart buffer
0,5 µl H2O

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

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

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Team:Marburg/Notebook:Mai

From 2013.igem.org

Revision as of 13:48, 3 October 2013

Notebook: May