Team:SDU-Denmark/Notebook

Notebook

Week 26

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we have done with the BioBrick K2018043 can be read in this protocol: Link to protocol (K2018043 - Cloning Basic part into iGEM standard plasmid psB1C3

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we have done with the BioBrick K2018044 can be read in this protocol: Link to protocol (K2018044 - Cloning Basic part into iGEM standard plasmid psB1C3)

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we had done with the BioBrick K1763003 can be read in this protocol: Link to protocol (K1763003 - Cloning Basic part into iGEM standard plasmid psB1C3)

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we had done with the BioBrick K1763004 can be read in this protocol: Link to protocol (K1763004 - Cloning Basic part into iGEM standard plasmid psB1C3)

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we had done with the BioBrick K1763009 can be read in this protocol: Link to protocol (K1763009 - Cloning Basic part into iGEM standard plasmid psB1C3)

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we had done with the BioBrick K1763010 can be read in this protocol: Link to protocol (K1763010 - Cloning Basic part into iGEM standard plasmid psB1C3)

The brick was ordered from IDT and received: The received gblock had a weight of 200 ng. and was resuspended in dH2O. 5 µl diluted gblock and 95 µl ddH2O to make a 20X dilution. What we had done with the BioBrick K1763012 can be read in this protocol: Link to protocol (K1763012 - Cloning Basic part into iGEM standard plasmid psB1C3)

Week 27

The BioBrick was ordered from IDT in a plasmid pUCIDT-AMP and after receiving it, it was resuspended to a final concentration of 0.1 µg/µl. After the resuspension, the plasmid was transformed into Top10 E. coli by following SOP0023_v01, with the following remarks: 2.5 µl plasmid was used instead of the normal 1 µl. What we had done with the BioBrick K2018011 can be read in this protocol: Link to protocol (K2018011 - Cloning composite part into iGEM standard plasmid), (Cloning Thuricin with DA silk-overhangs into pSB1C3), (Cloning Thuricin with DE silk-overhangs into pSB1C3), (Cloning Thuricin with EA silk-overhangs into pSB1C3), (Cloning Thuricin with ED silk-overhangs into pSB1C3) and (IMPACT Purification of K2018011)

Week 28

The BioBrick was ordered from IDT in a plasmid pUCIDT-AMP and after receiving it, it was resuspended to a final concentration of 0.1 µg/µl. After the resuspension, the plasmid was transformed into Top10 E. coli by following SOP0023_v01, with the following remarks: 2.5 µl plasmid was used instead of the normal 1 µl. What we had done with the BioBrickK2018010 can be read in this protocol: Link to protocol (K2018010 - Cloning composite part into iGEM standard plasmid) and (IMPACT Purification of K2018010)

The BioBrick containing the gene K2018011 was digested with PstI and XbaI overnight by using SOP0017_v01.

After digesting, the sample was ligated by following SOP0015_v01 with the standard iGEM plasmid, pSB1C3, overnight, and transformed into E.coli Top10.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 98 °C 2 min
2 34 cycles 98 °C 10 sec
    72 °C 15 sec
    72 °C >15 sec
3 Final extension 72 °C 5 min
4 Keep the sample cold 12 °C HOLD

After a colony PCR, SOP0021_v01, was run and verified on a gel, the following result was given: Our part is 259 bp and primer overhangs are 150 X 2 bp, which is, in total, 559 bp: A gel electrophoresis from cPCR showed a band around 600 bp, so we assumed that we had the correct part inserted into the iGEM standard plasmid.

What we had done with the BioBrick K2018011 can be read in this protocol: Link to protocol (K2018011 - Cloning composite part into iGEM standard plasmid), (Cloning Thuricin with DA silk-overhangs into pSB1C3), (Cloning Thuricin with DE silk-overhangs into pSB1C3), (Cloning Thuricin with EA silk-overhangs into pSB1C3), (Cloning Thuricin with ED silk-overhangs into pSB1C3) and (IMPACT Purification of K2018011)

The BioBrick was ordered from IDT in a plasmid pUCIDT-AMP and after receiving it, it was resuspended to a final concentration of 0.1 µg/µl. After the resuspension, the plasmid was transformed into Top10 E. coli by following SOP0023_v01, with the following remarks: 2.5 µl plasmid was used instead of the normal 1 µl. What we had done with the BioBrickK2018014 can be read in this protocol: Link to protocol (K2018014 - Cloning composite part into iGEM standard plasmid) and (IMPACT Purification of K2018014)

The BioBrick was ordered from IDT in a plasmid pUCIDT-AMP and after receiving it, it was resuspended to a final concentration of 0.1 µg/µl. After the resuspension, the plasmid was transformed into Top10 E. coli by following SOP0023_v01, with the following remarks: 2.5 µl plasmid was used instead of the normal 1 µl. What we had done with the BioBrickK2018019 can be read in this protocol: Link to protocol (K2018019 - Cloning composite part into iGEM standard plasmid) and (IMPACT Purification of K2018019)

Week 29

The BioBrick was first amplified by phusion PCR, SOP0010_v01, thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG47, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG47 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be observed .

T--SDU_Denmark_Notebook--2_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band, which was desired.

The second gel photo shows cutting of SB71 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrickK1763002 can be read in this protocol: Link to protocol K1763002 - Cloning Basic part into iGEM standard plasmid psB1C3) span>

The BioBrick was first amplified by phusion PCR, SOP0010_v01, and thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG48, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG48 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be observed .

T--SDU_Denmark_Notebook--2_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band.

The second gel photo shows cutting of SB73 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrickK1763003 can be read in this protocol: Link to protocol K1763003 - Cloning Basic part into iGEM standard plasmid psB1C3) span>

The BioBrick was first amplified by phusion PCR, SOP0010_v01, and thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG49, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG49 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be seen.

T--SDU_Denmark_Notebook--2_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band.

The second gel photo shows cutting of SB75 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrickK1763004 can be read in this protocol: Link to protocol K1763004 - Cloning Basic part into iGEM standard plasmid psB1C3) span>

The BioBrick was first amplified by phusion PCR, SOP0010_v01, and thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C >30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG56, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG56 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be observed .

T--SDU_Denmark_Notebook--2_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band.

The second gel photo shows cutting of SB77 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrickK1763009 can be read in this protocol: Link to protocol K1763009 - Cloning Basic part into iGEM standard plasmid psB1C3) span>

The BioBrick was first amplified by phusion PCR, SOP0010_v01, and thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C >30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG43, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG43 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be observed.

T--SDU_Denmark_Notebook--4_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band.

The second gel photo shows cutting of SB63 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrickK1763010 can be read in this protocol: Link to protocol K1763010 - Cloning Basic part into iGEM standard plasmid psB1C3) span>

The BioBrick was first amplified by phusion PCR, SOP0010_v01, and thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C >30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG44, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG44 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be observed .

T--SDU_Denmark_Notebook--4_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band.

The second gel photo shows cutting of SB65 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrickK1763011 can be read in this protocol: Link to protocol K1763011 - Cloning Basic part into iGEM standard plasmid psB1C3) span>

The BioBrick was first amplified by phusion PCR, SOP0010_v01, and thereafter digested with restrictions enzymes, following SOP0017_v01. The restriction enzymes were: PstI for 2 h and EcoRI for 30 min. After digesting, the sample was verified by running a gel electrophoresis.

pPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C >30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

In the next step, the vector was ligated with the insert, SG45, overnight by following SOP0015_v01, and thereafter it was transformed, following SOP0009_v01, into E.coli Top10. The ratio of the ligation is shown hereunder:

Ratio 1:2 1:5
Ligase Buffer 2 µl 2 µl
Ligase 1 µl 1 µl
SG45 2 µl 5 µl
SG58 1 µl 1 µl
ddH2O 14 µl 11 µl

To verify that the transformation and ligation succeeded, a colony PCR was run, SOP0021_v01.

cPCR program used:

Segment Step Temperature Duration
1 Initial denaturation 95 °C 3 min
2 34 cycles 95 °C 25 sec
    58 °C 25 sec
    72 °C 30 sec
3 Final extension 72 °C 1 min
4 Keep the sample cold 20 °C HOLD

The Gel Photo below shows the cPCR results. VR and VF2 primers adds a total of 300 bp, the gene is 119 bp which gives a total of DNA length of 419 bp. From this gel, the ligation of the gen incorporated into psB1C3 can be observed .

T--SDU_Denmark_Notebook--4_and_.png

Picture of cPCR of different colonies on the same plate (each gene had its own plate). All the colony PCR shows a band near the 4-500 bp band.

The second gel photo shows cutting of SB67 with Pst1 at 2 hours with 37 °C and EcoR1 at 30 minutes. Every second well had added restriction enzymes.If a band of approximately 160 bp could be seen on the gel, it was concluded as being successful and sent to sequencing.

T--SDU_Denmark_Notebook--3_and_.png

What we had done with the BioBrick K1763012 can be read in this protocol: Link to protocol K1763012 - Cloning Basic part into iGEM standard plasmid psB1C3) span>