Team:HokkaidoU Japan/Notebook/aggregation Week 12

Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2

To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI.
<bbr /> Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)

DNA solution (45~50 ng/ul)	27 ul
XbaI	1 ul
PstI	1 ul
10xM buffer	4 ul
DW	7 ul
Total	40 ul

Vector(ptet-pSB1A2)

DNA solution (about 20 ng/ul)	4 ul
SpeI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	12 ul
Total	20 ul

Number	Degree	Minute
1	37	120
2	60	15
3	4	HOLD

Number	Degree	Minute
1	37	120
2	70	20
3	4	HOLD

Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 15 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.

Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

Insert DNA (60~70 ng/ul)	4 ul
Vector DNA (20~30 ng/ul)	2 ul
Ligation Mighty Mix	6 ul
Total	12 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation of ligation ligation product into JM109.

1. Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).
5. Plated 300 ul of the culture onto first dish and spread.
6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated the plates at 37C for 17 hrs.

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3

We did colony PCR two times.

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	10 ul
Forward Primer(200bp down primer)	0.8 ul
Reverse Primer(ag43-f4 primer)	0.8 ul
DW	4.4 ul
Total	20 ul

Number	Degree	Second
1	95	120
2	95	30
3	53.0	30
4	72	60
5	72	60
6	4	HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls. Desired product is about 702bp.

Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once more.

Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3

To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI and pSB1C3 with EcoRI and SpeI.

Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)

DNA solution (about 35ng/ul)	41 ul
EcoRI	2 ul
SpeI	2 ul
10xH buffer	5 ul
Total	50 ul

Vector(pSB1C3)

DNA solution (about 30~40 ng/ul)	2 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	2 ul
DW	14 ul
Total	20 ul

Number	Degree	Minute
1	37	120
2	60	15
3	4	HOLD

Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 15 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3

Insert DNA	4 ul
Vector DNA	2 ul
Ligation Mighty Mix	6 ul
Total	12 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3

Transformation ligation product into JM109.

1. Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Incubated for 2 hrs to get the resistance to Chloramphenicol.
5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
6. Plated 300 ul of the culture onto first dish and spread.
7. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
8. Incubated the plates at 37C for 15 hrs.

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

Colony PCR to confirm whether pSTV28 vettor has pBAS-RBs-eCFP-RBs-Ag43-dT as insert or not.

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	10 ul
Forward Primer(200bp down primer)	0.8 ul
Reverse Primer(ag43-f4 primer)	0.8 ul
DW	4.4 ul
Total	20 ul

Number	Degree	Second
1	95	120
2	95	30
3	53.3	30
4	72	180
5	72	60
6	4	HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls. Desired product is about 702bp.

We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3 and pBAD-RBS-eCFP-RBS-Ag43-dI on pSB1C3

Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can anneal to a part of eCFP coding site and another can anneal to a part of Ag43 coding site.

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	10 ul
Forward Primer(FP-F primer)	0.8 ul
Reverse Primer(ag43-R primer)	0.8 ul
DW	4.4 ul
Total	20 ul

Number	Degree	Second
1	95	120
2	95	30
3	53.3	30
4	72	180
5	72	60
6	4	HOLD

Cycle:2~4 x 35

We used N1, N2 (DW only) as controls. Desired product is about 452 bp and 697 bp respectively.

We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.

Digestion of ptet-RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 (DNA solution eluted from colony No.10, 12 respectively in ) and ptet-pSB1A2

To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI. We prepared DNa solution derived from No. 10 and No. 12 colony selected selected by the result of colony PCR for 18th.

Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)

DNA solution (40 ng/ul)	20 ul
EcoRI	1 ul
PstI	1 ul
10xH buffer	4 ul
DW	4 ul
Total	30 ul

Insert No.12 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)

DNA solution (40 ng/ul)	20 ul
EcoRI	1 ul
PstI	1 ul
10xH buffer	4 ul
DW	4 ul
Total	30 ul

Vector(ptet-pSB1A2)

DNA solution (about 40 ng/ul)	2 ul
EcoRI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	14 ul
Total	20 ul

Number	Degree	Minute
1	37	120
2	60	15
3	4	HOLD

Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 15 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We decided to use No. 10 digestion product for ligation, and confirmed that the concentration of Insert DNA solution is 50 ng/ul and Vector DNA solution is about 20~30 ng/ul.

Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

Insert DNA (50 ng/ul)	4 ul
Vector DNA (20~30 ng/ul)	1.5 ul
DW	0.5 ul
Ligation Mighty Mix	6 ul
Total	12 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation of ligation product into JM109.

1. Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Incubated for 2 hrs to get the resistance to Chloramphenicol.
5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
6. Plated 300 ul of the culture onto first dish and spread.
7. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
8. Incubated the plates at 37C for 15 hrs.

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	10 ul
Forward Primer(X-phaB-F primer)	0.8 ul
Reverse Primer(PS-R primer)	0.8 ul
DW	4.4 ul
Total	20 ul

Number	Degree	Second
1	95	120
2	95	30
3	68.9	30
4	72	60
5	72	60
6	4	HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2(RBS-phaC-RBs-phaA-RBS-phaB-pSB1A2)as controls. Desired product is about 1500bp.

Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for plasmid extraction and No. 6, 12, 14 for storing at 4C.