Team:HokkaidoU Japan/Notebook/aggregation Week 12

(Difference between revisions)
 Revision as of 11:02, 26 September 2012 (view source)Slecat (Talk | contribs)← Older edit Revision as of 15:30, 26 September 2012 (view source)Slecat (Talk | contribs) Newer edit → Line 8: Line 8: ==Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2== ==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 & PstI, ptet-pSB1A2 with SpeI & PstI. + 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. Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2) Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2) {|class="hokkaidou-table-digestion" {|class="hokkaidou-table-digestion" |- |- - |DNA solution ( 45=50ng/ul) + |DNA solution (45~50 ng/ul) |27 ul |27 ul |- |- Line 148: Line 148: ==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2== ==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==

- Transformation of JM109. + Transformation of ligation ligation product in JM109. #Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice. #Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice. #Incubated on ice for 30 min. #Incubated on ice for 30 min. Line 235: Line 235: ==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3== ==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 & SpeI and pSB1C3 with EcoRI & SpeI. + 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) Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28) Line 371: Line 371: ==September 19th== ==September 19th==

Line 438: Line 438:

- Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can aneal to a part of eCFP coding site and another can aneal to a part of Ag43 coding site. + 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. {|class="hokkaidou-table-pcr-reagent" {|class="hokkaidou-table-pcr-reagent" |- |- Line 513: Line 513:

==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== ==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 & PstI, ptet-pSB1A2 with SpeI & PstI. We prepared DNa solution derived from No.10 and No.12 colony selected selected by the result of colony PCR for 18th. + 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) Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2) {|class="hokkaidou-table-digestion" {|class="hokkaidou-table-digestion" |- |- - |DNA solution ( 40 ng/ul) + |DNA solution (40 ng/ul) |20 ul |20 ul |- |- Line 541: Line 541: {|class="hokkaidou-table-digestion" {|class="hokkaidou-table-digestion" |- |- - |DNA solution ( 40 ng/ul) + |DNA solution (40 ng/ul) |20 ul |20 ul |- |- Line 618: Line 618: [[image:HokkaidoU2012 120919 Ethapre InsertNo10-InsertNo12-Vector.jpg|thumb|ethanol precipitation result]] [[image:HokkaidoU2012 120919 Ethapre InsertNo10-InsertNo12-Vector.jpg|thumb|ethanol precipitation result]] - 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. + 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.

Line 663: Line 663: ==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2== ==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==

- Transformation of JM109. + Transformation of ligation product in JM109. #Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice. #Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice. #Incubated on ice for 30 min. #Incubated on ice for 30 min. Line 743: Line 743: - Target products exist in almost all samples. We selected No.1,2 colony for incubation for mini-prep and No.6,12,14 for storing at 4C. + Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for mini-prep and No. 6, 12, 14 for storing at 4C.

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. 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 air drying at room temperature then added 5 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 in 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 hours.

September 18th

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.

Colony PCR result
Colony PCR result 2

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.

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

digestion result

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 air drying at room temperature then added 5 ul of DW.
ethanol precipitation result

 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 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 hours.

September 19th

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.

Colony PCR result

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

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.

Colony PCR result

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

digestion result

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 air drying at room temperature then added 5 ul of DW.
ethanol precipitation result

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 in 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 hours.

September 21th

 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.

Colony PCR result
Colony PCR result2

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