Team:Marburg/Notebook:June

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Notebook: June

03.06.2013

Digest
Investigator: Franzi
Aim: Construction of PNR-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.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

Ligation
Investigator: Franzi
Aim: Construction of PNR-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 PNR-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 LBCM, 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 (iBB9)

60 ng SPTP1 (iBB10)

50 ng SPTP2 (iBB11)

55 ng SP1 (iBB12)

50 ng SP2 (iBB13)

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 LBCM, oN 37°C.

Inoculation
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

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

04.06.2013

Miniprep
Investigator: Franzi
Aim: Construction of PfcpB-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 PfcpB-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-electrophoresis-image

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)

Lane   Content   Expectations
2+3   iBB4864 K1-2   2580 bp
4   empty  
5-7   iBB16 K3-5   650 bp

Digest
Investigator: Franzi
Aim: Construction of PfcpB-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.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

Ligation
Investigator: Franzi
Aim: Construction of PfcpB-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 PNR-test-vector and BioBricks → Inoculation of transformants.

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

05.06.2013

Miniprep
Investigator: Franzi
Aim: Construction of PNR-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 PNR-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-electrophoresis-image

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)

Lane   Content   Expectations
2-4   iBB9 K1-3   780 bp
5   empty  
6-8   iBB10 K1-3   228 bp
    pSB1C3: 2 kb

gel-electrophoresis-image

Lane   Content   Expectations
2-4   iBB11 K1-3   147 bp
5   empty  
6-8   iBB12 K1-3   123 bp
    pSB1C3: 2 kb

gel-electrophoresis-image

Lane   Content   Expectations
2-4   iBB13 K1-3   108 bp
5   empty  
6-8   iBB6315 K1-3   1200 bp
    pSB1C3: 2 kb

Transformation
Investigator: Franzi
Aim: Construction of PfcpB-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 LBCM (1C3)/LBamp (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 PfcpB-test-vector and antibody-vector → Inoculation of pSB1C3 iBB5416 and pSB1A3 iBB48647631 for miniprep.

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

07.06.2013

Miniprep
Investigator: Patrick
Aim: Construction of PfcpB-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 PfcpB-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-electrophoresis-image

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)

Lane   Content   Expectations
2-4   iBB5416 K1-3  

pSB1C3: 2070 bp

iBB5416: 1500 bp
5-8   iBB48647631 K1-4  

pSB1A3: 2150 bp

iBB48647631: 4157 bp

10.06.2013

Digest
Investigator: Lukas
Aim: Construction of PNR-test-vector, PfcpB-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.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

Ligation
Investigator: Christian
Aim: Construction of PNR-test-vector, PfcpB-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 PNR-test-vector, PfcpB-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 LBamp, oN 37°C.

12.06.2013

Inoculation
Investigator: Franzi
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

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

13.06.2013

Miniprep
Investigator: Franzi
Aim: Construction of PNR-test-vector, PfcpB-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 PNR-test-vector, PfcpB-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-electrophoresis-image

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)

Lane   Content   Expectations
upper:
2+3   iBB49 K3-4   1260 bp
4-7   iBB39 K1-4   1030 bp
lower:
2   iBB49647631   4157 bp
3-6   iBB496476315 K1-4   4404 bp
7+8   iBB49 K1-2   1260 bp
    Vector pSB1A3: 2150 bp

IBB48647631 and iBB486476315 probably positive, but better repeat or do a Colony-PCR.

16.06.2013

Digest
Investigator: Lukas
Aim: Construction of antibody-vector → Repeat of control digestion.
  • 1 µl DNA template
  • 0,3 µl EcoRI-HF
  • 0,3 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7,4 µl H2O

Templates: PSB1A3 iBB486476315 and pSB1A3 iBB48647631 K3 (comparison of size).

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

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2+3   iBB48647631   4157 bp
4-7   iBB486476315   TBA bp
    Vector pSB1A3: 2150 bp

Too much DNA used, will be repeated as 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 buffer
  • 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 into 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-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide 50 ml gel
  • 4 µl 2-log DNA ladder (New England Biolabs)

Lane   Content   Expectations
upper:
2   iBB6315   1200 + 2000 bp
3   iBB49   1200 + 2000 bp
4-7   iBB496315 K4-1   2400 + 2000 bp
lower:
2   iBB5416   1500 + 2000 bp
3   iBB39   1000 + 2000 bp
4-7   iBB395416 K1-4   2400 + 2000 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-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide 50 ml gel
  • 4 µl 2-log DNA ladder (New England Biolabs)

Lane   Content   Expectations
2-5   pSB1A3 iBB486476315 K1-4  

~ 6,000 bp for pSB1A3_486476315

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.

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 Pst/I
  • 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.

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-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide in 50 ml gel
  • 4 µl 2-log DNA ladder (New England Biolabs)

Lane   Content   Expectations
upper:
2-5   iBB395416 K1-4   2400 + 2000 bp
6   iBB39   1000 + 2000 bp
7   iBB6416   1400 + 2000 bp
lower:
2-6   iBB496315 K1-5   2400 + 2000 bp
7   iBB49   1200 + 2000 bp

All clones show a double band above 2000 bp.

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

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