Team:HokkaidoU Japan/Notebook/aggregation Week 4

From 2012.igem.org

(Difference between revisions)
Line 8: Line 8:
==Mini-prep==
==Mini-prep==
<p>
<p>
-
mini-prep for Ag43(7/17 cultivated colony resuspended and 7/20 cultivated colony resuspended). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.  
+
mini-prep for Ag43(7/17 cultivated colony resuspended and 7/20 cultivated colony resuspended). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.  
</p>
</p>
Line 30: Line 30:
==Gel extraction==
==Gel extraction==
<p>
<p>
-
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.  
+
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
</p>
</p>
==Ethanol precipitation==
==Ethanol precipitation==
<p>
<p>
Ethanol precipitation for digestion and gel extraction product.
Ethanol precipitation for digestion and gel extraction product.
-
#Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
+
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged in 15000 rpm, 10min at 4C.
-
#Remove supernatant and added 220ul of 70% ethanol.
+
#Remove supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged in 15000 rpm, 10 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 10ul of DW.  
+
#Remove supernatant and air drying in room temperature then added 10 ul of DW.  
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]]
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]]
Line 56: Line 56:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |5ul
+
   |5 ul
   |-
   |-
   |PstI
   |PstI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |2ul
+
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |12ul
+
   |12 ul
   |-
   |-
   |Total
   |Total
-
   |20ul
+
   |20 ul
   |}
   |}
Line 77: Line 77:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |12ul
+
   |12 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |XbaI
   |XbaI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |2ul
+
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |4ul
+
   |4 ul
   |-
   |-
   |Total
   |Total
-
   |20ul
+
   |20 ul
   |}
   |}
Line 107: Line 107:
==Gel extraction==
==Gel extraction==
<p>
<p>
-
Gel ectraction of Ag43-dt digestio result.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.
+
Gel ectraction of Ag43-dt digestio result.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
</p>
</p>
Line 118: Line 118:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |8ul
+
   |8 ul
   |-
   |-
   |HindIII
   |HindIII
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
Line 146: Line 146:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |3ul
+
   |3 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |DW
   |DW
-
   |5ul
+
   |5 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
Line 169: Line 169:
<p>
<p>
Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI).
Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI).
-
We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.  
+
We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
</p>
</p>
Line 175: Line 175:
<p>
<p>
Ethanol precipitation of digestion and gel extraction products.
Ethanol precipitation of digestion and gel extraction products.
-
#Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
+
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged in 15000 rpm, 10min at 4C.
-
#Remove supernatant and added 220ul of 70% ethanol.
+
#Remove supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000rpm, 5min at 4C.
+
#Centrifuged in 15000 rpm, 5 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 5ul of DW.  
+
#Remove supernatant and air drying in room temperature then added 5 ul of DW.  
</p>
</p>
Line 189: Line 189:
|-
|-
|pT7-RBS on pSB1K3
|pT7-RBS on pSB1K3
-
|2ul
+
|2 ul
|-
|-
|Ag43-dT
|Ag43-dT
-
|2ul
+
|2 ul
|-
|-
|DW
|DW
-
|1ul
+
|1 ul
|-
|-
|Ligation Mighty Mix(TAKARA)
|Ligation Mighty Mix(TAKARA)
-
|5ul
+
|5 ul
|-
|-
|Total
|Total
-
|10ul
+
|10 ul
|}
|}
Line 231: Line 231:
<p>
<p>
Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).
Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).
-
#Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
+
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
-
#Incubated on ice for 30min.
+
#Incubated on ice for 30 min.
-
#Added 200ul of LB then incubated the cells for 2 hrs at 37C.
+
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
#Prepared and Labeled two petri dishes with LBK.
#Prepared and Labeled two petri dishes with LBK.
-
#Plate 200ul of the transformation onto first dish and spread.
+
#Plate 200 ul of the transformation onto first dish and spread.
-
#Added 450ul of LB to 50ul of the transformation and plated 200ul of it onto second dish and spread.  
+
#Added 450 ul of LB to 50 ul of the transformation and plated 200 ul of it onto second dish and spread.  
-
#Incubated the plates at 37C for over 30hrs.
+
#Incubated the plates at 37C for over 30 hrs.
There were no colony on the plates.
There were no colony on the plates.
Line 247: Line 247:
#Placed TBE agarose gel in Electrophoresis chamber.
#Placed TBE agarose gel in Electrophoresis chamber.
#Added 1/2X TBE buffer to Electrophoresis chamber.
#Added 1/2X TBE buffer to Electrophoresis chamber.
-
#Added 5ul of Etbr and ran at 100V in 30min.  
+
#Added 5 ul of Etbr and ran at 100 V in 30 min.  
#Load 1kb DNA ladder and each samples.
#Load 1kb DNA ladder and each samples.
-
#Ran at 100V in 30min.
+
#Ran at 100 V in 30 min.
[[image:HokkaidoU2012 120726 digestion ligation.jpg|thumb|Digestion and Ligation results]]
[[image:HokkaidoU2012 120726 digestion ligation.jpg|thumb|Digestion and Ligation results]]
Line 264: Line 264:
<p>
<p>
Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).
Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).
-
#Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
+
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
-
#Incubated on ice for 30min.
+
#Incubated on ice for 30 min.
-
#Added 600ul of LB then incubated the cells for 2 hrs at 37C.
+
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
#Prepared and Labeled three petri dishes with LBK X2 and LBC.
#Prepared and Labeled three petri dishes with LBK X2 and LBC.
-
#Plate 300ul of the transformation onto LBK dish and spread.
+
#Plate 300 ul of the transformation onto LBK dish and spread.
-
#Added 900ul of LB to 100ul of the transformation and plated 300ul of it onto LBC dish and LBK dish then spread.  
+
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBC dish and LBK dish then spread.  
-
#Incubated the plates at 37C for 17hrs 30min.
+
#Incubated the plates at 37C for 17 hrs and 30 min.
</p>
</p>
Line 280: Line 280:
|-
|-
|pT7-RBS on pSB1K3
|pT7-RBS on pSB1K3
-
|2ul
+
|2 ul
|-
|-
|Ag43-dT
|Ag43-dT
-
|2ul
+
|2 ul
|-
|-
|DW
|DW
-
|1ul
+
|1 ul
|-
|-
|Ligation Mighty Mix(TAKARA)
|Ligation Mighty Mix(TAKARA)
-
|5ul
+
|5 ul
|-
|-
|Total
|Total
-
|10ul
+
|10 ul
|}
|}
Line 326: Line 326:
<p>
<p>
Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.
Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.
-
#Added 2ml of LBC into culture tubes.
+
#Added 2 ml of LBC into culture tubes.
#Resuspended 5 colonies.
#Resuspended 5 colonies.
-
#Incubated the tubes at 30C for 22hrs.
+
#Incubated the tubes at 30C for 22 hrs.
</p>
</p>
Line 339: Line 339:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |3ul
+
   |3 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |DW
   |DW
-
   |5ul
+
   |5 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}  
   |}  
Line 358: Line 358:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |8ul
+
   |8 ul
   |-
   |-
   |HindIII
   |HindIII
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
-
   |1ul
+
   |1 ul
   |-
   |-
   |Total
   |Total
-
   |10ul
+
   |10 ul
   |}
   |}
Line 378: Line 378:
==Gel extraction==
==Gel extraction==
<p>
<p>
-
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.  
+
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
</p>
</p>
==Ethanol precipitation==
==Ethanol precipitation==
<p>
<p>
Ethanol precipitation for digestion and gel extraction product.
Ethanol precipitation for digestion and gel extraction product.
-
#Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
+
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged in 15000 rpm, 10 min at 4C.
-
#Remove supernatant and added 220ul of 70% ethanol.
+
#Remove supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged in 15000 rpm, 10 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 10ul of DW.  
+
#Remove supernatant and air drying in room temperature then added 10 ul of DW.  
</p>
</p>
Line 397: Line 397:
|-
|-
|pT7-RBS on pSB1K3
|pT7-RBS on pSB1K3
-
|2ul
+
|2 ul
|-
|-
|Ag43-dT
|Ag43-dT
-
|2ul
+
|2 ul
|-
|-
|DW
|DW
-
|1ul
+
|1 ul
|-
|-
|Ligation Mighty Mix(TAKARA)
|Ligation Mighty Mix(TAKARA)
-
|5ul
+
|5 ul
|-
|-
|Total
|Total
-
|10ul
+
|10 ul
|}
|}
Line 437: Line 437:
==mini-prep==
==mini-prep==
<p>
<p>
-
mini-prep for five pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.  
+
mini-prep for five pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.  
[[image:HokkaidoU2012_120729_pt7-rbs-ag43-dt.jpg|thumb|mini-prep products]]
[[image:HokkaidoU2012_120729_pt7-rbs-ag43-dt.jpg|thumb|mini-prep products]]

Revision as of 10:21, 6 August 2012

Contents

July 23th

Mini-prep

mini-prep for Ag43(7/17 cultivated colony resuspended and 7/20 cultivated colony resuspended). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.


July 24th

Electrophoresis

Electrophoresis for Ag43(mini-preped above) and Ag43 digestion results(digested with EcoRI and SpeI)

Mini-prep result
Digestion result

In this digestion result, we knew that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use in digestion.

Gel extraction

Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 10min 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.
Ethanol precipitation result

In this result, we estimated that the concentration of ethanol precipitation product is about 40ng/ul.

Digestion

Digestion to confirm how many PstI cutting sites are in K346007(Ag43 coading) and Ag43-dT complex digestion with SpeI and XbaI. Ag43 PstI

DNA solution 5 ul
PstI 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul


Ag43-dT SpeI and XbaI

DNA solution 12 ul
SpeI 1 ul
XbaI 1 ul
10xH buffer 2 ul
DW 4 ul
Total 20 ul

Electrophoresis

Electrophoresis for digestion results.

Ag43 d+(P) Digestion result
Ag43-dT d+(X&S) Digestion result

In this result, we found that there are 6 PstI cutting sites in K346007(Ag43).

Gel extraction

Gel ectraction of Ag43-dt digestio result.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Digestion

Digestion for Ag43-dT and pSB1AK3 mixture(each DNA fragment is about 3kbp) with HindIII to digest pSB1AK3 into about two 1.5kbp fragments.

DNA solution 8 ul
HindIII 1 ul
10xM buffer 1 ul
Total 10 ul


July 25th

Digestion

Digestion of pT7-RBS on pSB1K3 cutting with SpeI.

DNA solution 3 ul
SpeI 1 ul
10xH buffer 1 ul
DW 5 ul
Total 10 ul
Digestion result(Ag43-dT and pT7-RBS)

We were confirmed that pAB1AK3 was digested into 1.3k and 1.8k bp fragments by HindIII. But there are a little doubt SpeI wasn't work because the band of pT7-RBS on pSB1K3 of d- and d+ existed same bp area.

Gel extraction

Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI). We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation of digestion and gel extraction products.

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

Ligation

Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2 ul
Ag43-dT 2 ul
DW 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold



July 26th

Transformation

Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).

  1. Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Plate 200 ul of the transformation onto first dish and spread.
  6. Added 450 ul of LB to 50 ul of the transformation and plated 200 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for over 30 hrs.
There were no colony on the plates.

Electrophoresis

Electrophoresis of digestion and ligation products.

  1. Placed TBE agarose gel in Electrophoresis chamber.
  2. Added 1/2X TBE buffer to Electrophoresis chamber.
  3. Added 5 ul of Etbr and ran at 100 V in 30 min.
  4. Load 1kb DNA ladder and each samples.
  5. Ran at 100 V in 30 min.
Digestion and Ligation results

There are no band in the lane of ligation products. But if digestion products didn't ligate, there are two bands of digestion products would exist in the lane.


July 27th

Transformation

Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).

  1. Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled three petri dishes with LBK X2 and LBC.
  5. Plate 300 ul of the transformation onto LBK dish and spread.
  6. Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBC dish and LBK dish then spread.
  7. Incubated the plates at 37C for 17 hrs and 30 min.

Ligation

Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2 ul
Ag43-dT 2 ul
DW 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold



July 28th

Transformation result left:LBC center:LBK right:LBK

There were many colonies in LBC! We guess we mistook pT7-RBS on pSB1C3 for pT7-RBS on pSB1K3.

Liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.

  1. Added 2 ml of LBC into culture tubes.
  2. Resuspended 5 colonies.
  3. Incubated the tubes at 30C for 22 hrs.

Digestion

Digestion of pT7-RBS on pSB1K3 with SpeI and Ag43-dT on pSB1AK3(cut with SpeI & XbaI) with HindIII. pT7-RBS on pSB1K3

DNA solution 3 ul
SpeI 1 ul
10xH buffer 1 ul
DW 5 ul
Total 10 ul

Ag43-dT on pSB1AK3(cut with SpeI & XbaI)

DNA solution 8 ul
HindIII 1 ul
10xM buffer 1 ul
Total 10 ul

Digestioned at 37C in 2hrs.

digestion result

This results confirmed that pSB1AK3 was successfully digested into fragments(1.3K and 1.8K bp), but we weren't confirmed whether pT7-RBS on pSB1K3 was digested or not.

Gel extraction

Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 10 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.

Ligation

Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2 ul
Ag43-dT 2 ul
DW 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold


July 29th

mini-prep

mini-prep for five pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

mini-prep products

Digestion

We need to check the DNA is really pT7-RBS-Ag43-dT or not. mini-prep products(pT7-RBS-Ag43-dT) PstI

DNA solution 4 ul
PstI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


mini-prep products(pT7-RBS-Ag43-dT) EcoR1 and Spe1

DNA solution 4 ul
EcoR1 1 ul
Spe1 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul

Electrophoresis

Electrophoresis for digestion results.

Digestion result

I could not understand what is happend. I tried it again, but the result was the same.


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