Team:HokkaidoU Japan/Notebook/aggregation Week 8

From 2012.igem.org

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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.
 +
 +
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{{Team:HokkaidoU_Japan/footer}}
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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.

  1. Picked up one colony.
  2. 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.

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.


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).

  1. Added 2 ml of LBK(LBC) into culture tubes.
  2. Resuspended 1 colonies(Resuspended pre-cultivated 200ul of LB and colony solution).
  3. 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.

  1. 2 ml of liquid culture divided two culture. (made two 1 ml culture)
  2. Added 1 ml LBK in one culture as negative control.
  3. Added 900 ul LBK and 100 ul 20% L-arabinose.
  4. Incubated at 37C 130rpm for 2hours and 30minutes.
  5. Placed tubes on the table at 30minutes.
</div>


</div> </div> </div>

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