Team:HokkaidoU Japan/Notebook/aggregation Week 9
From 2012.igem.org
(Difference between revisions)
(27th colony pcr, incubation) |
(28th) |
||
Line 72: | Line 72: | ||
- | ==Incubation for mini-prep== | + | ==Incubation for mini-prep of pT7-RBS-Ag43-dT on pSB1C3== |
<p> | <p> | ||
Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium. | Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium. | ||
Line 78: | Line 78: | ||
#Re-suspended 2 colonies (No.1 and No.2 respectively). | #Re-suspended 2 colonies (No.1 and No.2 respectively). | ||
#Incubated at 37C for 15 hrs. | #Incubated at 37C for 15 hrs. | ||
+ | </p> | ||
+ | |||
+ | ==Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2= | ||
+ | <p> | ||
+ | #Mixed 1 ul DNA to 50 ul of thawed competent cells on ice. | ||
+ | #Incubated on ice for 30 min. | ||
+ | #Mixed 350 ul of LB. | ||
+ | #Prepared and Labeled two plastic plates with LBC. | ||
+ | #Plated 300 ul of the culture onto first dish and spread. | ||
+ | #Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread. | ||
+ | #Incubated the plates at 37C for 15 hours. | ||
+ | </p> | ||
+ | |||
+ | ==PCR of RBS-YFP-dT== | ||
+ | <p> | ||
+ | Amplified the construct by 100bp-up-EX primer and 200bp-down-PS primer. | ||
+ | Mixed PCR solutions. | ||
+ | {|class="hokkaidou-table-pcr-reagent" | ||
+ | |Solution | ||
+ | |Volume(ul) | ||
+ | |- | ||
+ | |DNA | ||
+ | |1 | ||
+ | |- | ||
+ | |100bp-up-EX | ||
+ | |1 | ||
+ | |- | ||
+ | |200bp-down-PS | ||
+ | |1 | ||
+ | |- | ||
+ | |MgSO4 | ||
+ | |3 | ||
+ | |- | ||
+ | |dNTP | ||
+ | |5 | ||
+ | |- | ||
+ | |10x KOD-Plus-Neo Buffer | ||
+ | |5 | ||
+ | |- | ||
+ | |KOD-Plus-Neo | ||
+ | |1 | ||
+ | |- | ||
+ | |DW | ||
+ | |33 | ||
+ | |- | ||
+ | |Total | ||
+ | |50 | ||
+ | |} | ||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |94 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |98 | ||
+ | |10 | ||
+ | |- | ||
+ | |3 | ||
+ | |58.2 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |68 | ||
+ | |60 | ||
+ | |- | ||
+ | |5 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 35 | ||
+ | |||
+ | </p> | ||
+ | </div></div> | ||
+ | |||
+ | |||
+ | <div class="hokkaidou-notebook-daily"> | ||
+ | ==August 28th== | ||
+ | <div> | ||
+ | ==mini-prep of pT7-RBS-Ag43-dT on pSB1C3== | ||
+ | Mini-prep of pT7-RBS-Ag43-dT on pSB1C3 | ||
+ | <p> | ||
+ | |||
+ | [[image:|thumb|mini-prep result]] | ||
+ | |||
</p> | </p> | ||
</div></div> | </div></div> |
Revision as of 16:48, 28 August 2012
Contents |
August 27th
Colony PCR
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not.
DNA solution | 4 ul |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(ag43-f4 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 | 53.0 | 30 |
4 | 72 | 60 |
5 | 72 | 60 |
6 | 4 | HOLD |
Cycle:2~4 x 35
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls. Desired product is about 695bp.
[[image:|thumb|Colony PCR result]]
Incubation for mini-prep of pT7-RBS-Ag43-dT on pSB1C3
Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium.
- Prepared 2 ml LBC into culture tubes.
- Re-suspended 2 colonies (No.1 and No.2 respectively).
- Incubated at 37C for 15 hrs.
=Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2
- Mixed 1 ul DNA to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Mixed 350 ul of LB.
- Prepared and Labeled two plastic plates with LBC.
- Plated 300 ul of the culture onto first dish and spread.
- Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
- Incubated the plates at 37C for 15 hours.
PCR of RBS-YFP-dT
Amplified the construct by 100bp-up-EX primer and 200bp-down-PS primer. Mixed PCR solutions.
Solution | Volume(ul) |
DNA | 1 |
100bp-up-EX | 1 |
200bp-down-PS | 1 |
MgSO4 | 3 |
dNTP | 5 |
10x KOD-Plus-Neo Buffer | 5 |
KOD-Plus-Neo | 1 |
DW | 33 |
Total | 50 |
Number | Degree | Second |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 58.2 | 30 |
4 | 68 | 60 |
5 | 4 | HOLD |
Cycle:2~4 x 35
August 28th
mini-prep of pT7-RBS-Ag43-dT on pSB1C3
Mini-prep of pT7-RBS-Ag43-dT on pSB1C3
[[image:|thumb|mini-prep result]]