Team:Marburg/Notebook:Mai

From 2013.igem.org

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Revision as of 18:09, 3 October 2013

Notebook: May

02.05.2013

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

5 colonies of pSB1C3 iBB8 inoculated in 5 ml LBcm each, 2 colonies of 2xNR HC + LC inoculated in 5 ml LBamp 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  

For gel see digest below.

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-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    
2-6   Control digestion iBB8 K1-5    
7   Test-PCR 2xNR HC+LC    

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 LBamp, 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 LBamp 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-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    
2+3   pSB1A3 J04450 K1    
4   empty    
5+6   pSB1A3 J04450 K2    

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.

They were 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 LBamp, 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 LBamp, 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 LBamp, 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.

The samples were 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 LBamp, 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 LBamp, 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-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
2-5   iBB64 K1-4   700 bp
6+7   iBB76 K1-2   1000 bp

gel-electrophoresis-image

Lane   Content   Expectations
2-3   iBB76 K3-4   1000 bp
4-7   iBB31 K1-4   700 bp

gel-electrophoresis-image

Lane   Content   Expectations
2-5   iBB48 K1-4   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.

They were 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 LBcm, 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 LBcm, 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 LBcm, 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 LBcm, 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-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
2-5   iBB7631 K1-4  

Vector: 2150 bp

Insert: 1610 bp

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

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

27.05.2013

Digest
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-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.

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

Ligation
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-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 PNR-test-vector and PfcpB-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 LBamp, 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-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    
2   pSB1C3 J04450 (EcoRI/PstI cut)    

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-electrophoresis-image

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)

Lane   Content   Expectations
2   iBB9   780 bp
3   iBB10   228 bp
4   iBB11   147 bp
5   iBB12   123 bp
6   iBB13   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 LBCM, oN 37°C.

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

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

29.05.2013

Miniprep
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-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 PNR-test-vector and PfcpB-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-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   iBB63 K1-2   500 bp
4+5   iBB15 K1-2   700 bp

gel-electrophoresis-image

Lane   Content   Expectations
2+3   iBB54 K1-2   750 bp
4+5   iBB16 K1-2   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-electrophoresis-image

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)

Lane   Content   Expectations
upper:
2+3   iBB9   780 bp
4   empty  
5+6   iBB10   228 bp
7   empty  
8   iBB11   147 bp
lower:
2+3   iBB12   123 bp
4   empty  
5+6   iBB13   108 bp
7   empty  
8   iBB11   147 bp

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.

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

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