Team:HokkaidoU Japan/Notebook/aggregation Week 10
From 2012.igem.org
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</p> | </p> | ||
+ | |||
+ | |||
+ | [[image:|thumb|digestion result]] | ||
+ | |||
+ | |||
+ | We confirmed that ptet-RBS-eYFP-dT-pSB1A2 was partially digested and pSTV28 would be successfully digested we think. But we couldn't confirm whether Ag43-dT-pSB1AK3 was digested or not. | ||
+ | We did a gel-extraction of these digested DNA and got 50ul solution of each DNA. | ||
</div></div> | </div></div> | ||
+ | |||
+ | <div class="hokkaidou-notebook-daily"> | ||
+ | ==September 6th== | ||
+ | <div> | ||
+ | ==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28== | ||
+ | <p> | ||
+ | #Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol. | ||
+ | #Centrifuged in 14000 rpm, 30 min at 4C. | ||
+ | #Remove supernatant and added 220 ul of 70% ethanol. | ||
+ | #Centrifuged in 15000 rpm, 15 min at 4C. | ||
+ | #Remove supernatant and air drying in room temperature then added 5 ul of DW. | ||
+ | |||
+ | |||
+ | [[image:|thumb|ethanol precipitation result]] | ||
+ | We confirmed that the concentration of Insert DNA solution is 10~20 ng/ul and Vector DNA solution is 30~40 ng/ul. | ||
+ | </p> | ||
+ | |||
+ | ==Ligation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28== | ||
+ | <p> | ||
+ | {|class="hokkaidou-table-ligation" | ||
+ | |- | ||
+ | |Vector DNA (30~40 ng/ul) | ||
+ | |1.5 ul | ||
+ | |- | ||
+ | |Insert DNA (10~20 ng/ul) | ||
+ | |4 ul | ||
+ | |- | ||
+ | |Ligation Mighty Mix | ||
+ | |6 ul | ||
+ | |- | ||
+ | |DW | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |12 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | Ligation reaction time was in detail below. | ||
+ | |||
+ | {|class="hokkaidou-table-ligation" | ||
+ | |- | ||
+ | |Degree | ||
+ | |Minute | ||
+ | |- | ||
+ | |16 | ||
+ | |30 | ||
+ | |- | ||
+ | |65 | ||
+ | |10 | ||
+ | |- | ||
+ | |4 | ||
+ | |Hold | ||
+ | |} | ||
+ | |||
+ | </p> | ||
+ | |||
+ | ==Transformation of ptet-RBS-eYFP-dT-pSTV28== | ||
+ | <p> | ||
+ | #Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice. | ||
+ | #Incubated on ice for 30 min. | ||
+ | #Mixed 350 ul of LB. | ||
+ | #Incubated for 2 hrs to get the resistance to Chloramphenicol. | ||
+ | #Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (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 25 hours. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | ==Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2== | ||
+ | <p> | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-reagent" | ||
+ | |- | ||
+ | |DNA solution | ||
+ | |4 ul | ||
+ | |- | ||
+ | |Kapa-Taq(Taq polymerase) | ||
+ | |5 ul | ||
+ | |- | ||
+ | |Forward Primer(pbad-f2 primer) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Reverse Primer(PS-R down primer) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |10 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |95 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |95 | ||
+ | |30 | ||
+ | |- | ||
+ | |3 | ||
+ | |53.3 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |5 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |6 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 35 | ||
+ | |||
+ | We used N1 (DW only) as controls. | ||
+ | Desired product is about 900bp. | ||
+ | |||
+ | [[image:|thumb|Colony PCR result]] | ||
+ | |||
+ | |||
+ | We thought that almost all of ligated DNA successfully ligated to our desired construct. We selected No.1 colony for incubation and store No.2,3 and 4 colony liquid medium at 4C. | ||
+ | </p> | ||
+ | |||
+ | </div></div> | ||
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --> | <!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --> | ||
</div> | </div> | ||
<br style="line-height: 0; clear: both;" /> | <br style="line-height: 0; clear: both;" /> | ||
{{Team:HokkaidoU_Japan/footer}} | {{Team:HokkaidoU_Japan/footer}} |
Revision as of 03:53, 6 September 2012
September 3rd
Colony PCR of eCFP-RBS-pSB1A2
DNA solution | 4 ul |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(EX-F primer) | 0.5 ul |
Reverse Primer(PS-R down primer) | 0.5 ul |
Total | 10 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 (ptetR-RBS-eCFP-dT-pSB1A2) as controls. Desired product is about 776bp.
We confirmed that about 70% of ligated DNA formed our desired construct. We selected No.2 and 5 colony for incubation and store No.7 and 8 colony mixture at 4C.
Incubation of eCFP-RBS-pSB1A2 for mini-prep
- Prepared 2 ml LBA into culture tubes.
- Re-suspended 2 colony mixture (No.2 and No.5 respectively).
- Incubated at 37C for hrs.
Estimation of concentration of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2
No.2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2. [[image:|thumb|electrophoresis result]] We estimated the concentration of eCFP-RBS-pSB1A2 is 30 ng/ul and pBAD-RBS-pSB1A2 is 50ng/ul.
Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2
To make a construct of pBAD-RBS-eCFP-RBS-pSB1A2, we digested eCFP-RBS-pSB1A2 with XbaI & PstI and pBAD-RBS-pSB1A2 with SpeI & PstI. And we digested eCFP-pSB1A2 with XbaI & SpeI as a control for confirmation of the ability to digest. Insert (eCFP-RBS-pSB1A2)
DNA solution ( 30ng/ul) | 22 ul |
XbaI | 1 ul |
PstI | 1 ul |
10xM buffer | 3 ul |
DW | 3 ul |
Total | 30 ul |
Vector(pBAD-RBS-pSB1A2)
DNA solution ( 50ng/ul) | 3 ul |
SpeI | 1 ul |
PstI | 1 ul |
10xH buffer | 2 ul |
DW | 13 ul |
Total | 20 ul |
control (eCFP-pSB1A2)
DNA solution (30 ng/ul) | 5 ul |
XbaI | 1 ul |
SpeI | 1 ul |
10xM buffer | 2 ul |
DW | 11 ul |
Total | 20 ul |
Number | Degree | Minute |
1 | 37 | 120 |
2 | 60 | 15 |
3 | 4 | HOLD |
[[image:|thumb|digestion result]]
From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked.
September 4th
Ethanol precipitation of digestion products (eCFP-RBS and pBAD-RBS-pSB1A2) and estimation of concentration
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged in 15000 rpm, 15 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged in 15000 rpm, 10 min at 4C.
- Remove supernatant and air drying in room temperature then added 5 ul of DW.
Ligation of eCFP-RBS and pBAD-RBS-pSB1A2
Vector DNA (50 ng/ul) | 1.5 ul |
Insert DNA (20 ng/ul) | 4 ul |
Ligation Mighty Mix | 6 ul |
DW | 0.5 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-pSB1A2
- Mixed 2 ul pBAD-RBS-eCFP-RBS-pSB1A2 ligation product 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 LB plate medium which contained appropriate antibiotics (LBA).
- 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 25 hours.
Digestion of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28
To make a construct of ptet-RBS-eYFP-dT-pSTV28, we digested ptet-RBS-eYFP-dT-pSB1A2 with EcoRI & PstI and pSTV28 with EcoRI & PstI. And we digested Ag43-dT-pSB1AK3 with HindIII. Insert (ptet-RBS-eYFP-dT-pSB1A2)
DNA solution ( 30ng/ul) | 20 ul |
EcoRI | 1 ul |
PstI | 1 ul |
10xH buffer | 3 ul |
DW | 5 ul |
Total | 30 ul |
Vector(pSTV28)
DNA solution ( 50ng/ul) | 3 ul |
EcoRI | 1 ul |
PstI | 1 ul |
10xH buffer | 2 ul |
DW | 13 ul |
Total | 20 ul |
Ag43-dT-pSB1AK3
DNA solution (30 ng/ul) | 30 ul |
HindIII | 2 ul |
10xM buffer | 5 ul |
DW | 13 ul |
Total | 50 ul |
Number | Degree | Minute |
1 | 37 | 120 |
2 | 60 | 15 |
3 | 4 | HOLD |
[[image:|thumb|digestion result]]
We confirmed that ptet-RBS-eYFP-dT-pSB1A2 was partially digested and pSTV28 would be successfully digested we think. But we couldn't confirm whether Ag43-dT-pSB1AK3 was digested or not.
We did a gel-extraction of these digested DNA and got 50ul solution of each DNA.
September 6th
Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged in 14000 rpm, 30 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged in 15000 rpm, 15 min at 4C.
- Remove supernatant and air drying in room temperature then added 5 ul of DW.
Ligation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28
Vector DNA (30~40 ng/ul) | 1.5 ul |
Insert DNA (10~20 ng/ul) | 4 ul |
Ligation Mighty Mix | 6 ul |
DW | 0.5 ul |
Total | 12 ul |
Ligation reaction time was in detail below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Transformation of ptet-RBS-eYFP-dT-pSTV28
- Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Mixed 350 ul of LB.
- Incubated for 2 hrs to get the resistance to Chloramphenicol.
- Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (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 25 hours.
Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2
DNA solution | 4 ul |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(pbad-f2 primer) | 0.5 ul |
Reverse Primer(PS-R down primer) | 0.5 ul |
Total | 10 ul |
Number | Degree | Second |
1 | 95 | 120 |
2 | 95 | 30 |
3 | 53.3 | 30 |
4 | 72 | 60 |
5 | 72 | 60 |
6 | 4 | HOLD |
Cycle:2~4 x 35
We used N1 (DW only) as controls. Desired product is about 900bp.
[[image:|thumb|Colony PCR result]]
We thought that almost all of ligated DNA successfully ligated to our desired construct. We selected No.1 colony for incubation and store No.2,3 and 4 colony liquid medium at 4C.