Team:HokkaidoU Japan/Notebook/aggregation Week 8
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+ | <div class="hokkaidou-notebook-daily"> | ||
+ | ==August 20th== | ||
+ | <div> | ||
+ | ==Single colony isolation== | ||
+ | <p> | ||
+ | Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3. | ||
+ | #Picked up one colony. | ||
+ | #Cultivation on LBK(dt,RBS,T7) and LBK(pLacI-RBS-Ag43) in hours. | ||
+ | </p> | ||
+ | ==Colony PCR== | ||
+ | <p> | ||
+ | Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-reagent" | ||
+ | |- | ||
+ | |DNA solution | ||
+ | |4 ul | ||
+ | |- | ||
+ | |Kapa-Taq(Taq polymerase) | ||
+ | |5 ul | ||
+ | |- | ||
+ | |Forward Primer(100bp up primer) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Reverse Primer(200bp down primer) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |10 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |95 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |95 | ||
+ | |30 | ||
+ | |- | ||
+ | |3 | ||
+ | |53.2 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |5 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |6 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 35 | ||
+ | |||
+ | We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls. | ||
+ | Desired product is about 300~400bp. | ||
+ | |||
+ | [[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]] | ||
+ | |||
+ | The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evapolated because of our mistake. We selected No.4 and 5 colony for liquid culture. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | ==PCR== | ||
+ | <p> | ||
+ | PCR of BBa_I13453 (pBAD only part, it is not contain araC,). | ||
+ | |||
+ | {|class="hokkaidou-table-pcr-reagent" | ||
+ | |- | ||
+ | |DNA solution | ||
+ | |1 ul | ||
+ | |- | ||
+ | |KOD-Plus-NEO(Taq polymerase) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |dNTP | ||
+ | |5 ul | ||
+ | |- | ||
+ | |MgSO4 | ||
+ | |3 ul | ||
+ | |- | ||
+ | |KOD-Plus-NEO Buffer | ||
+ | |5 ul | ||
+ | |- | ||
+ | |Forward Primer(Ag43-f4 primer: 10 uM) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |Reverse Primer(PS-R primer: 10 uM) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |DW | ||
+ | |33 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |50 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |94 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |98 | ||
+ | |10 | ||
+ | |- | ||
+ | |3 | ||
+ | |58 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |68 | ||
+ | |30 | ||
+ | |- | ||
+ | |5 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 45 | ||
+ | |||
+ | |||
+ | |||
+ | [[image:|thumb|PCR result]] | ||
+ | |||
+ | </p> | ||
+ | ==liquid culture== | ||
+ | <p> | ||
+ | Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR). | ||
+ | #Added 2 ml of LBK(LBC) into culture tubes. | ||
+ | #Resuspended 1 colonies(Resuspended pre-cultivated 200ul of LB and colony solution). | ||
+ | #Incubated the tubes at 37C for 16 hours. | ||
+ | </p> | ||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
+ | <div class="hokkaidou-notebook-daily"> | ||
+ | ==August 21th== | ||
+ | <div> | ||
+ | ==PCR== | ||
+ | <p> | ||
+ | PCR of pBAD(containing araC)-RBS. | ||
+ | And, we checked the plasmid which we did mini-prep at August 18th is pBAD-RBS on pSB1A3 or not. | ||
+ | |||
+ | {|class="hokkaidou-table-pcr-reagent" | ||
+ | |- | ||
+ | |DNA solution | ||
+ | |1 ul | ||
+ | |- | ||
+ | |KOD-Plus-NEO(Taq polymerase) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |dNTP | ||
+ | |5 ul | ||
+ | |- | ||
+ | |MgSO4 | ||
+ | |3 ul | ||
+ | |- | ||
+ | |KOD-Plus-NEO Buffer | ||
+ | |5 ul | ||
+ | |- | ||
+ | |Forward Primer(Ag43-f4 primer: 10 uM) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |Reverse Primer(PS-R primer: 10 uM) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |DW | ||
+ | |33 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |50 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |94 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |98 | ||
+ | |10 | ||
+ | |- | ||
+ | |3 | ||
+ | |58 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |68 | ||
+ | |30 | ||
+ | |- | ||
+ | |5 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 45 | ||
+ | |||
+ | |||
+ | |||
+ | [[image:|thumb|PCR result]] | ||
+ | |||
+ | |||
+ | ==Aggregation check== | ||
+ | |||
+ | we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK. | ||
+ | We checked the construct by induction of L-arabinose after 16 hours incubate. | ||
+ | |||
+ | #2 ml of liquid culture divided two culture. (made two 1 ml culture) | ||
+ | #Added 1 ml LBK in one culture as negative control. | ||
+ | #Added 900 ul LBK and 100 ul 20% L-arabinose. | ||
+ | #Incubated at 37C 130rpm for 2hours and 30minutes. | ||
+ | #Placed tubes on the table at 30minutes. | ||
+ | |||
+ | </div><div> | ||
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Revision as of 08:22, 21 August 2012
Contents |
August 20th
Single colony isolation
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.
- Picked up one colony.
- Cultivation on LBK(dt,RBS,T7) and LBK(pLacI-RBS-Ag43) in hours.
Colony PCR
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not.
DNA solution | 4 ul |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(100bp up primer) | 0.5 ul |
Reverse Primer(200bp down primer) | 0.5 ul |
Total | 10 ul |
Number | Degree | Second |
1 | 95 | 120 |
2 | 95 | 30 |
3 | 53.2 | 30 |
4 | 72 | 60 |
5 | 72 | 60 |
6 | 4 | HOLD |
Cycle:2~4 x 35
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls. Desired product is about 300~400bp.
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evapolated because of our mistake. We selected No.4 and 5 colony for liquid culture.
PCR
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).
DNA solution | 1 ul |
KOD-Plus-NEO(Taq polymerase) | 1 ul |
dNTP | 5 ul |
MgSO4 | 3 ul |
KOD-Plus-NEO Buffer | 5 ul |
Forward Primer(Ag43-f4 primer: 10 uM) | 1 ul |
Reverse Primer(PS-R primer: 10 uM) | 1 ul |
DW | 33 ul |
Total | 50 ul |
Number | Degree | Second |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 58 | 30 |
4 | 68 | 30 |
5 | 4 | HOLD |
Cycle:2~4 x 45
[[image:|thumb|PCR result]]
liquid culture
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).
- Added 2 ml of LBK(LBC) into culture tubes.
- Resuspended 1 colonies(Resuspended pre-cultivated 200ul of LB and colony solution).
- Incubated the tubes at 37C for 16 hours.
August 21th
PCR
PCR of pBAD(containing araC)-RBS. And, we checked the plasmid which we did mini-prep at August 18th is pBAD-RBS on pSB1A3 or not.
DNA solution | 1 ul |
KOD-Plus-NEO(Taq polymerase) | 1 ul |
dNTP | 5 ul |
MgSO4 | 3 ul |
KOD-Plus-NEO Buffer | 5 ul |
Forward Primer(Ag43-f4 primer: 10 uM) | 1 ul |
Reverse Primer(PS-R primer: 10 uM) | 1 ul |
DW | 33 ul |
Total | 50 ul |
Number | Degree | Second |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 58 | 30 |
4 | 68 | 30 |
5 | 4 | HOLD |
Cycle:2~4 x 45
[[image:|thumb|PCR result]]
Aggregation check
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK. We checked the construct by induction of L-arabinose after 16 hours incubate.
- 2 ml of liquid culture divided two culture. (made two 1 ml culture)
- Added 1 ml LBK in one culture as negative control.
- Added 900 ul LBK and 100 ul 20% L-arabinose.
- Incubated at 37C 130rpm for 2hours and 30minutes.
- Placed tubes on the table at 30minutes.
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