Team:HokkaidoU Japan/Notebook/aggregation Week 11

From 2012.igem.org

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We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No.1,2 for incubation for mini-prep and No.3,4 for stock at 4C.
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No.1,2 for incubation for mini-prep and No.3,4 for stock at 4C.
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</p>
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==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for mini-prep==
 +
<p>
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#Prepared 2 ml LBC into culture tubes.
 +
#Re-suspended 2 colony mixture (No.1 and No.2 respectively).
 +
#Incubated at 37C for 14 hrs.
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</p>
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</div></div>
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<div class="hokkaidou-notebook-daily">
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==September 14th==
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<div>
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==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==
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<p>
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To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI & PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI & PstI.
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 +
Insert (RBS-phaB-RBS-eYFP-dT)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution ( 30ng/ul)
 +
  |21 ul
 +
  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |3 ul
 +
  |-
 +
  |DW
 +
  |4 ul
 +
  |-
 +
  |Total
 +
  |30 ul
 +
  |}
 +
 +
 +
Vector(RBS-phaC-RBS-phaA-pSB1A2)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (about 30~40 ng/ul)
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  |6 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |10 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|Number
 +
|Degree
 +
|Minute
 +
|-
 +
|1
 +
|37
 +
|180
 +
|-
 +
|2
 +
|60
 +
|15
 +
|-
 +
|3
 +
|4
 +
|HOLD
 +
|}
 +
 +
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|Number
 +
|Degree
 +
|Minute
 +
|-
 +
|1
 +
|37
 +
|120
 +
|-
 +
|2
 +
|70
 +
|20
 +
|-
 +
|3
 +
|4
 +
|HOLD
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|}
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 +
 +
[[image:|thumb|digestion result]]
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 +
We suppose the Insert DNA (RBS-phaB-RBS-eYFP-dT) has contained Vector-dimer DNA as contamination. But desired DNA fragment (about 1600 bp) bond also appeared in the result of migration. We picked up the fragment and extracted from migrated TBE gel.
</p>
</p>
</div></div>
</div></div>

Revision as of 07:05, 15 September 2012

Contents

September 10th

Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 with EcoRI & SpeI, Ag43-dT-pSB1AK3 (previously digested with HindIII) with XbaI & NotI and pSTV28 with EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 with XbaI & SpeI as a control for confirmation of the ability of restriction enzyme. Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)

DNA solution ( 35ng/ul) 17 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 3 ul
DW 8 ul
Total 30 ul


Insert2 (Ag43-dT-pSB1AK3)

DNA solution ( 35ng/ul) 25 ul
XbaI 1 ul
NotI 1 ul
10xK buffer 1.5 ul
100xBSA 0.3ul
DW 1.5 ul
Total 30 ul



Vector(pSTV28)

DNA solution ( 15ng/ul) 9 ul
EcoRI 1 ul
NotI 1 ul
10xH buffer 2 ul
10xBSA 0.2ul
DW 7 ul
Total 20 ul


control (pT7-RBS-pSB1C3)

DNA solution (30~40 ng/ul) 10 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 2 ul
DW 6 ul
Total 20 ul


Number Degree Minute
1 37 120
2 70 20
3 4 HOLD


digestion result

Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 14000 rpm, 30 min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 15 min at 4C.
  5. Remove supernatant and air drying in room temperature then added 5 ul of DW.
ethanol precipitation result

We confirmed that the concentration of Insert1 DNA solution is 50 ng/ul, Insert2 DNA solution is 10~15 ng/ul and Vector DNA solution is 10 ng/ul.

Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28

Vector DNA (10 ng/ul) 4 ul
Insert1 DNA (50 ng/ul) 2 ul
Insert2 DNA (10~15 ng/ul) 4 ul
Ligation Mighty Mix 10 ul
Total 20 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold

Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

  1. Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 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 14 hours.


September 11th

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 noticed that this step was actually 68.9 degree after reaction.

We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls. Desired product is about 542bp.

[[image:|thumb|Colony PCR result]]


Because of mis-setting the PCR program, we failed to amplify desired DNA fragment so we retried the colony PCR in same reagent, correct PCR machine program.

[[image:|thumb|Colony PCR result]]


We noticed that the ligated DNA contains at least Ag43-dT-BioBrick_Suffix complex. We selected No.2,4 for incubation for mini-prep and No.5,6 for Aggregation check (see details below).


September 12th

Aggregation check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

Aggregation check for No.5,6 colonies selected by colony PCR in 11th.

  1. Prepared 5 ml LBA into glass tubes.
  2. Re-suspended 2 colony mixture (No.2 and No.5 respectively).
  3. Incubated at 37C for hrs.

Digestion of RBS-phaB-pSB1A2 as Vector and RBS-eYFP-dT as Insert

To make a construct of RBS-phaB-RBS-eYFP-dT-pSB1A2, we digested RBS-phaB-pSB1A2 with SpeI & PstI, RBS-eYFP-dT with XbaI & PstI.


Insert (RBS-eYFP-dT)

DNA solution ( 40ng/ul) 25 ul
XbaI 1 ul
NotI 1 ul
10xK buffer 1.5 ul
100xBSA 0.3ul
DW 1.5 ul
Total 30 ul



Vector(RBS-phaB-pSB1A2)

DNA solution ( 35ng/ul) 6 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 10 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD


[[image:|thumb|digestion result]] </p>

Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 15 min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 10 min at 4C.
  5. Remove supernatant and air drying in room temperature then added 10 ul of DW.
File:.jpg
ethanol precipitation result

We confirmed that the concentration of Insert DNA solution is 40 ng/ul and Vector DNA solution is 30 ng/ul.

Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28

Vector DNA (40 ng/ul) 3 ul
Insert DNA (30 ng/ul) 3 ul
Ligation Mighty Mix 6 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold


September 13th

Transformation of RBS-phaB-RBS-eYFP-dT-pSB1A2

  1. Mixed 2 ul ligation product to 50 ul of thawed competent cells (JM109) 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 14 hours.

Aggregation Check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

  1. Mixed 25 ul Arabinose solution (20 %) to 5 ml LBC in glass tube.
  2. Incubated for 24 hrs at 37C.
Result: The construct has aggregated not forming large cluster but small dot cluster, and the supernatant has muddiness.

Colony PCR of RBS-phaB-RBS-eYFP-dT-pSB1A2

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 10 ul
Forward Primer(pbad-f2 primer: 10 ng/ul) 0.8 ul
Reverse Primer(PS-R down primer: 10 ng/ul) 0.8 ul
DW 4.4 ul
Total 20 ul


Number Degree Second
1 95 120
2 95 30
3 64.4 30
4 72 180
5 72 120
6 4 HOLD

Cycle:2~4 x 35


We used N1 (DW only) and N2(RBS-phaB-pSB1A2)as controls. Desired product is about 1800~2000bp.

[[image:|thumb|Colony PCR result]]


We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No.1,2 for incubation for mini-prep and No.3,4 for stock at 4C.


Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for mini-prep

  1. Prepared 2 ml LBC into culture tubes.
  2. Re-suspended 2 colony mixture (No.1 and No.2 respectively).
  3. Incubated at 37C for 14 hrs.


September 14th

Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI & PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI & PstI. Insert (RBS-phaB-RBS-eYFP-dT)

DNA solution ( 30ng/ul) 21 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 3 ul
DW 4 ul
Total 30 ul


Vector(RBS-phaC-RBS-phaA-pSB1A2)

DNA solution (about 30~40 ng/ul) 6 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 10 ul
Total 20 ul


Number Degree Minute
1 37 180
2 60 15
3 4 HOLD


Number Degree Minute
1 37 120
2 70 20
3 4 HOLD


[[image:|thumb|digestion result]]

We suppose the Insert DNA (RBS-phaB-RBS-eYFP-dT) has contained Vector-dimer DNA as contamination. But desired DNA fragment (about 1600 bp) bond also appeared in the result of migration. We picked up the fragment and extracted from migrated TBE gel.