From 2013.igem.org

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Revision as of 20:53, 1 October 2013

Notebook: June

Anmerkung

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03.06.2013

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-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:

Digestion of pSB1A3 iBB1+5 with XbaI and PstI-HF

Digestion of pSB1A3 iBB6+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.

Ligation

Investigator: Franzi

Aim: Construction of P_NR-test-vector → Ligation of two combined 2 BioBricks in pSB1C3.

45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
140 ng iBB6+3 (EcoRI/PstI-cut)
150 ng iBB1+5 (EcoRI/PstI-cut)
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 P_NR-test-vector → Transformation of combined 4 BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples were 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.

Ligation

Investigator: Franzi

Aim: Construction of BioBricks → Ligation of single BioBricks into pSB1C3.

45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
X ng insert
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl H2O

Samples (all EcoRI/PstI-cut):

120 ng eGFP

60 ng SPTP1

50 ng SPTP2

55 ng SP1

50 ng SP2

The samples were incubated for 3 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of BioBricks → Transformation of single BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples were 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_fcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

3 colonies of pSB1A3 iBB1+6 inoculated in 5 ml LB_amp, 4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LB_CM, oN 37°C.

04.06.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Miniprep of pSB1A3 iBB1+6 and pSB1C3 iBB4864.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).

Digest

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Control digestion of pSB1A3 iBB1+6 and pSB1C3 iBB4864.

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 2 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: 2 kb
iBB4864: 2580 bp
iBB1+6: 650 bp

All positive.

Digest

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Digestion of combined 2 BioBricks in pSB1A3 and combined 4 BioBricks in pSB1C3.

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

Samples:

Digestion of pSB1C3 iBB7631 K4 and pSB1A3 iBB16 K4 with XbaI and PstI-HF

Digestion of pSB1C3 iBB4864 K1 and pSB1A3 iBB54 K2 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_fcpB-test-vector and antibody-vector → Ligation of two combined 2 BioBricks into pSB1C3 and two combined 4 BioBricks into pSB1A3.

Antibody:

80 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
160 ng iBB7631
190 ng iBB4864
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl H2O

Test-vector:

45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
150 ng iBB54
120 ng iBB16
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl H2O

Both samples were incubated oN at 16° C.

Inoculation

Investigator: Franzi

Aim: Construction of P_NR-test-vector and BioBricks → Inoculation of transformants.

Transformations (03.06.13) inoculated in 5 ml LB_CM, oN 37°C.

05.06.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_NR-test-vector and BioBricks → Miniprep of single BioBricks and pSB1C3 iBB6315.

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

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector and BioBricks → Control digestion of single BioBricks and pSB1C3 iBB6315.

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 µl H2O

The samples were incubated for 1,5 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

pSB1C3: 2 kb
iBB9: 780 bp
iBB10: 228 bp
iBB11: 147 bp
iBB12: 123 bp
iBB13: 108 bp
iBB6315: 1200 bp

Problems with the TBE-buffer %rarr; Longer time with lower volt.

Transformation

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Transformation of combined 4 BioBricks in pSB1C3 and combined 8 BioBricks in pSB1A3 into E. coli DH5α.

Ligations (04.06.13) were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min (1C3)/40 min (1A3) 37°C + 900 µl LB), plated on LB_CM (1C3)/LB_amp (1A3), oN 37°C.

Sequencing

Investigator: Franzi

Aim: Construction of BioBricks → Examination of sequencing results of various constructs.

iBB9 K1 fw - AGB000K 263

iBB9 K1 rv - AGB000K 264

iBB9 K2 fw - AGB000K 265

iBB9 K2 rv - AGB000K 266

iBB10 K1 fw - AGB000K 267

iBB10 K2 fw - AGB000K 268

iBB11 K1 fw - AGB000K 269

iBB11 K2 fw - AGB000K 270

iBB12 K1 fw - AGB000K 271

iBB12 K2 fw - AGB000K 272

iBB13 K1 fw - AGB000K 273

iBB13 K2 fw - AGB000K 274

all correct, except iBB10 G→T pos.30, iBB11 T→C pos.24. Probably template mutated.

06.06.2013

Inoculation

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Inoculation of pSB1C3 iBB5416 and pSB1A3 iBB48647631 for miniprep.

3 colonies of pSB1C3 iBB5416 inoculated in 5 ml LB_CM, 4 colonies of pSB1A3 iBB48647631 inoculated in 5 ml LB_amp, oN 37°C.

07.06.2013

Miniprep

Investigator: Patrick

Aim: Construction of P_fcpB-test-vector and antibody-vector → Miniprep of pSB1C3 iBB5416 and pSB1A3 iBB48647631.

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

Digest

Investigator: Patrick

Aim: Construction of P_fcpB-test-vector and antibody-vector → Control digestion of pSB1C3 iBB5416 and pSB1A3 iBB48647631.

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 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

pSB1C3: 2070 bp
iBB5416: 1500 bp
pSB1A3: 2150 bp
iBB48647631: 4157 bp

All positive.

10.06.2013

Digest

Investigator: Lukas

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Digestion of pSB1A3 iBB48647631 and single BioBricks.

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

Samples:

Digestion of 1,5 µl pSB1A3 iBB48647631 with SpeI-HF and PstI-HF

Digestion of 5 µl pSB1C3 iBB5 and iBB9 with XbaI and PstI-HF

Digestion of 4 µl pSB1C3 iBB3 and iBB4 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: Christian

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Ligation of pSB1A3 iBB48647631 and single BioBricks in pSB1A3.

Ligation of pSB1A3 iBB48647631 with iBB5, pSB1A3 iBB48647631 with iBB3 and iBB9, pSB1A3 iBB48647631 with iBB4 and iBB9; oN 16°C

11.06.2013

Transformation

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector %rarr; Transformation of ligations.

Transformation of the ligations into E. coli DH5α (30 min ice, 60 sec 42°C, 25 min 37°C + 900 µl LB), plated on LB_amp, oN 37°C.

12.06.2013

Inoculation

Investigator: Franzi

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

4 colonies of each transformation inoculated in 4 ml LB_amp, oN 37°C.

13.06.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Miniprep of transformants.

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

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Control digestion the minipreps and pSB1A3 iBB48647631 K3 (comparison of size).

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 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

pSB1A3: 2150 bp
iBB48647631: 4157 bp
iBB486476315: 4404 bp
iBB49: 1260 bp
iBB39: 1030 bp

For better separation another 30 min.

Probably positive, but better repeat or do a Colony-PCR.

17.06.2013

Digest

Investigator: Alex

Aim: Digest of pSB1A3_3+9 and pSB1A3_4+9 with SpeI and PstI, and corresponding inserts pSB1C3_5+4+1+6 and pSB1C3_6+3+1+5 with EcoRI and PstI.

1 µl DNA
0.3 µl Enzyme 1 (SpeI or EcoRI, resp.)
0.3 µl Enzyme 2 (PstI)
1 µl Cut Smart
7.4 µl ddH2O

The samples were incubated for 1.5 h at 37° C.

Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (QIAGEN).

Ligation, Transformation, Plasmid prep

Investigator: Alex

Aim: Assemble the combined bricks 5416 and 6315 in their appropriate vectors pSB1A3_39 and pSB1A3_49, resp.

Insert und plasmid were transformed using a 1:2 ratio. Samples were incubated ON at RT.
Ligation product were transformed into chemical competent DH5α cells (30 min ice, 90 sec 42°C, 45 min 37°C + 900 µl LB).
Transformed cells were spread out on LB Amp culture plates.
Incubation ON at RT.
4 clones were picked for overnight cultures.
A plasmid preparation was carried out the next day.

22.06.2013

Test digest

Investigator: Alex

Aim: Digest of pSB1A3_395416 and pSB1A3_496315 with EcoRI and PstI.

1 µl Plasmid DNA
0.3 µl EcoRI
0.3 µL PstI
1 µl Cut Smart
7.4 µl ddH2O

The samples were incubated for 2 h at 37° C.

Gel electrophoresis

Gel substances

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

Expectations (upper gel)

Lane 2: 1,200 + 2,000 bp
Lane 3: 1,200 + 2,000 bp
Lane 4-7: 2,400 + 2,000 bp

Expectations (lower gel)

Lane 2: 1,500 + 2,000 bp
Lane 3: 1,000 + 2,000 bp
Lane 4-7: 2,400 + 2,000 bp

We didn’t receive all expected fragments. All expected fragments in lanes 4-7 were missing. Ligation failed :(

24.06.2013

PCR

Investigator: Alex

Aim: Check right assembly of biobrick BBa_K1071005 in the plasmid for antibody production

Volume	Reagent	Temp (°C)	Time
1 µl	pSB1A3_486476315	95	3 min
0.5 µl	Primer fwd	95	30 sec
0.5 µl	Primer rev	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
add 25 µL	ddH2O

Gel electrophoresis

Gel substances

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

Expectations

Lane 1: ~ 6,000 bp for pSB1A3_486476315
Lane 2: 280 bp for BBa_K1071005

Each sample contained 2 bands, that means we got all expected bands and it could be assumed that the insertion of BBa_K1071005 was successful.

25.06.2013

Digest

Investigator: Dominik

Aim: Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 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

Expectations

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

Worked as expected.

26.06.2013

Digest

Investigator: Dominik

Aim: Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9

1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)
0.5 µl EcoRI
0.5 µl SpeI
2 µl CutSmart
12 µl ddH2O

1 µl Plasmid (pSB1C3-iBB9)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
12 µl ddH2O

1 µl Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
12 µl ddH2O

The samples were incubated for 1.5 h at 37° C.

Gel electrophoresis

Gel substances

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

Expectations

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 iBB10+iBB9, iBB11+iBB9, iBB12+iBB9 and iBB13+iBB9 into the vector pSB1A3.

2 µl vector DNA (pSB1A3)
2 µl insert 1 DNA (iBB9)
2 µl insert 2 DNA (iBB10, iBB11, iBB12 or iBB13)
2 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase
2 µl ddH2O

The samples were incubated overnight at room temperature.

27.06.2013

Transformation

Investigator: Dominik

Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

Inoculation

Investigator: Dominik

Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

Digest

Investigator: Alex

Aim: Digest of pSB1A3-iBB395416 and pSB1A3-iBB496315 with EcoRI and PstI.

1 µl Plasmid DNA
0.3 µl EcoRI
0.3 µl PstI
1 µl Cut Smart
7.4 µl ddH2O

The samples were incubated for 2 h at 37° C.

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 2-5: 2,400 + 2,000 bp
Lane 6: 1,000 + 2,000 bp
Lane 7: 1,400 + 2,000 bp

Expectations (lower gel)

Lane 2-7: 2,400 + 2,000 bp
Lane 8: 1,200 + 2,000 bp

All clones show a double band above 2,000 bp.

We received all expected fragments. The success of the ligation should be double-checked by means of PCR.

@@ Line 1,140: / Line 1,140: @@
 					</p>
 					<p>
-						<span class="exp">Expactations</span>
+						<span class="exp">Expectations</span>
 						<ul class="exp">
 							<li>Lane 1: 5300 kbp</li>
@@ Line 1,222: / Line 1,222: @@
 					</p>
 					<p>
-						<span class="exp">Expactations</span>
+						<span class="exp">Expectations</span>
 						<ul class="exp">
 							<li>Lane 1: 5300 kbp</li>

Team:Marburg/Notebook:June

From 2013.igem.org

Revision as of 20:53, 1 October 2013

Notebook: June