Team:HokkaidoU Japan/Notebook/aggregation Week 1

From 2012.igem.org

(Difference between revisions)
(Incubation of mini-prep and glycerol stock)
(Digestion of I719005, B0034 and pSB1K3)
 
(16 intermediate revisions not shown)
Line 4: Line 4:
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi -->
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 2nd==
+
===July 2nd===
-
<div>
+
<div class="hokkaidou-section">
On your mark...
On your mark...
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 3rd===
-
==July 3rd==
+
<div class="hokkaidou-section">
-
<div>
+
Get set...
Get set...
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 4th===
-
==July 4th==
+
<div class="hokkaidou-section">
-
<div>
+
====Transformation====
-
===Transformation===
+
Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) into DH5&alpha;
-
<p>
+
#Mixed 1 ul DNA solution with DH5&alpha; competent cells and incubated on ice for 30 min.
-
Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) to DH5&alpha;
+
-
#Mixed 1 ul DNA solution with DH5&alpha; competent cells and incubated on ice in 30 min.
+
#Plated them on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). ]
#Plated them on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). ]
To gain chloramphenicol resistances transformed cell solution was incubated for 2 hrs.
To gain chloramphenicol resistances transformed cell solution was incubated for 2 hrs.
pT7 plate was incubated for 21 hrs and Others for 20 hrs.
pT7 plate was incubated for 21 hrs and Others for 20 hrs.
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 5th===
-
==July 5th==
+
<div class="hokkaidou-section">
-
<div>
+
====Transformation====
-
===Transformation===
+
'''K346007(Ag43) failed to grow on LBC plate.'''
-
<p>
+
Transformation of K346007(Ag43) into DH5&alpha;.
-
'''K346007(Ag43) failed to produce colonies on LBC plate.'''
+
-
 
+
-
 
+
-
Transformation of K346007(Ag43) to DH5&alpha;.
+
#Mixed 1 ul DNA solution with DH5&alpha; competent cells and incubated on ice for 30 min.
#Mixed 1 ul DNA solution with DH5&alpha; competent cells and incubated on ice for 30 min.
-
#To gain chrolamphenicol resistance solution was pre-cultivated for 2 hrs.
+
#To gain chrolamphenicol resistance solution was pre-incubated for 2 hrs.
#Incubated on LBC for 21 hrs.
#Incubated on LBC for 21 hrs.
-
</p>
 
-
===Single colony isolation===
+
====Single colony isolation====
-
<p>
+
Performed single colony isolation of BBa_B0015, B0034, I179005 and K542009.
Performed single colony isolation of BBa_B0015, B0034, I179005 and K542009.
#Incubated the plates for 14 hrs and 30 mins  
#Incubated the plates for 14 hrs and 30 mins  
Line 55: Line 42:
After consulting part page we found out that
After consulting part page we found out that
'''BBa_K542009 was Ag43 basic part, not composite! And the part didn't have Biobrick suffix.'''
'''BBa_K542009 was Ag43 basic part, not composite! And the part didn't have Biobrick suffix.'''
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 6th===
-
==July 6th==
+
<div class="hokkaidou-section">
-
<div>
+
====Incubation for plasmid extraction and glycerol stock====
-
===Incubation for mini-prep and glycerol stock===
+
-
<p>
+
Incubated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) solution.
Incubated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) solution.
#Picked up two colonies from each plate.
#Picked up two colonies from each plate.
-
#First colony was re-suspended in 1 ml LB (A and C respectively) for glycerol stock. Second colony was re-suspended in 2 ml LB(A and C respectively) for mini-prep.  
+
#First colony was re-suspended in 1 ml LB (A and C respectively) for glycerol stock. Second colony was re-suspended in 2 ml LB(A and C respectively) for plasmid extraction.  
#Incubated for 16 hrs.
#Incubated for 16 hrs.
-
</p>
 
-
===Single colony isolation===
+
====Single colony isolation====
-
<p>
+
#Single colony isolation of K346007(Ag43) on LBC.  
#Single colony isolation of K346007(Ag43) on LBC.  
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 7th===
-
==July 7th==
+
<div class="hokkaidou-section">
-
<div>
+
====Incubation of plasmid extraction and glycerol stock====
-
===Incubation of mini-prep and glycerol stock===
+
-
<p>
+
Incubation of (Ag43) in LBC liquid medium.
Incubation of (Ag43) in LBC liquid medium.
-
#Two colonies were re-suspended in 2 ml LBC. We incubated them at 38C.<br />
+
Two colonies were re-suspended in 2 ml LBC. We incubated them at 38C.<br />
'''One of the tubes was incubated for 8 hrs. It's for glycerol stock.'''
'''One of the tubes was incubated for 8 hrs. It's for glycerol stock.'''
-
</p>
 
-
===3A assembly===
+
====3A assembly!!!====
-
<p>
+
Assembled pT7, RBS and pSB1C3 by 3A assembly.
Assembled pT7, RBS and pSB1C3 by 3A assembly.
-
This 3A assembly is our first try!
+
This is our first try!
-
</p>
+
-
===mini-prep===
+
====Plasmid extraction====
-
<p>
+
#Plasmid extraction of dT,RBS,pT7 and pLacI-RBS-Ag43. We used FastGene Plasmid Mini Kit(Nippon Gene)
-
#mini-prep of dT,RBS,pT7 and pLacI-RBS-Ag43. We used FastGene Plasmid Mini Kit(Nippon Gene)
+
#We got 50 ul of DNA solutions.
-
#Got 50 ul of DNA solutions.
+
-
</p>
+
-
===Glycerol stock===
+
====Glycerol stock====
-
<p>
+
Glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.
-
glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.
+
#Parts written above were incubated in 1 ml of LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) for 16 hrs 30 min.
-
#Parts written above were cultivated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) 1 ml in 16 hrs 30 min.
+
#Add glycerol and Freeze at -80C
#Add glycerol and Freeze at -80C
-
</p>
 
-
===Electrophoresis===
+
====Estimation of plasmid concentration====
-
<p>
+
[[Image:120707_I719005_B0034_B0015_K542009_mini-prep_umeuchi.jpg|thumb|Erectrophoresis result]]
[[Image:120707_I719005_B0034_B0015_K542009_mini-prep_umeuchi.jpg|thumb|Erectrophoresis result]]
-
Electrophoresis to predict concentration of mini-prep products(dT,RBS,pT7 and pLacI-RBS-Ag43).
+
Electrophoresis to estimate the concentration of plasmid extraction products(dT,RBS,pT7 and pLacI-RBS-Ag43).
#Used 1% agarose gel.
#Used 1% agarose gel.
-
#Added EtBr into TBE buffer and started Pre-migration.
+
#Added EtBr to TBE buffer and soaked the gel by applying current.
-
#Migrated '''1.2 ul of DNA solutions (1 ul is mini-prep products and 0.2 ul is Loading Dye)''' in 35 min.
+
#Ran '''1.2 ul of DNA solutions (1 ul is plasmid extraction product and 0.2 ul is Loading Dye)''' for 35 min.
-
#Took a photograph of 1% agarose gel that finished electrophoresis.
+
#Took a photograph of 1% agarose gel.
-
</p>
+
-
 
+
-
===Digestion===
+
-
<p>
+
-
Digestion of I719005, B0034 and pSB1K3
+
-
Digestion recipe
+
====Digestion of I719005, B0034 and pSB1K3====
 +
Digestion reaction
-
All parts were reacted in 30ul digestion mix.
+
All parts were digested in 30 ul reaction solution.
*I719005(40 ng/ul)
*I719005(40 ng/ul)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
Line 184: Line 149:
|13 ul
|13 ul
|}
|}
-
</p>
+
 
-
===Ethanol precipitation===
+
====Ethanol precipitation of Digestion products====
-
<p>
+
Concentrating the DNA solution which and removing restriction enzymes.
-
For rising concentration of DNA solution which use for Ligation and removing restriction enzyme.
+
#Added 3 ul of NaoAc, 1.5 ul of glycogen and 75 ul of 100% ethanol.
#Added 3 ul of NaoAc, 1.5 ul of glycogen and 75 ul of 100% ethanol.
-
#Centrifuged in 14000 rpm, 30 min at 4C.
+
#Centrifuged at 14000 rpm, 30 min at 4C.
-
#Remove supernatant and added 220ul of 70% ethanol.
+
#Removed supernatant and added 220ul of 70% ethanol.
-
#Centrifuged in 15000 rpm, 15 min at 4C.
+
#Centrifuged at 15000 rpm, 15 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 10 ul of DW.
+
#Removed supernatant and air dried at room temperature. Dissolved in 10 ul of DW.
-
</p>
+
-
===Ligation===
+
====Ligation (3A Assembly)====
-
<p>
+
3A assembly requires Ligation reaction total volume to be 25 ul.
-
All DNA solutions were digested.
+
-
3A assembly protocol required Ligation reaction should be in total 25 ul solution.
+
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
|-
|-
Line 220: Line 181:
|}
|}
-
Ligation reaction recipe was written below.
+
 
 +
Incubation of ligation reaction.
{|class="hokkaidou-table-pcr-time"
{|class="hokkaidou-table-pcr-time"
Line 236: Line 198:
|Hold
|Hold
|}
|}
-
<br />
+
 
 +
 
ligation was finished.  
ligation was finished.  
-
But now pm10:00.  2.5hrs needs for doing transformation. Transformation would finish at am:0:30.
+
But now is 10 pm.  2.5hrs are needed for transformation. Transformation would be finished at 0:30 am.
Withdraw!!!!
Withdraw!!!!
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 8th===
-
==July 8th==
+
<div class="hokkaidou-section">
-
<div>
+
*(pT7 + RBS)
*(pT7 + RBS)
-
===Transformation===
+
====Transformation====
-
<p>
+
Transformation for pT7+RBS+pSB1K3
Transformation for pT7+RBS+pSB1K3
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.
-
#Stood on ice in 30 min.
+
#Stood on ice for 30 min.
#Added 600 ul of LB to transformed DH5&alpha; solution.
#Added 600 ul of LB to transformed DH5&alpha; solution.
-
#Pre-cultivate in 2 hrs
+
#Pre-incubated for 2 hrs at 37C.
#Splead 300 ul of LB&DH5&alpha; solution to LBK.
#Splead 300 ul of LB&DH5&alpha; solution to LBK.
-
#Cultivated
+
#Incubated for 19 hrs.
-
</p> 
+
 
-
*K346007(Ag43)
+
===Plasmid extraction===
-
===mini-prep===
+
Plasmid extraction for Liquid culture product of K346007(Ag43)
-
<p>
+
-
mini-prep for Liquid culture product of K346007(Ag43)
+
#Used FastGene Plasmid Mini Kit(Nippon Genetics)
#Used FastGene Plasmid Mini Kit(Nippon Genetics)
#Elutioned in 50 ul
#Elutioned in 50 ul
-
#'''First we eluted in colection tube. then moved in microcentrifuge tube.'''  
+
#'''First we eluted in collection tube. then moved in micro-centrifuge tube.'''  
-
</p>
+
 
-
===Erectrophoresis===
+
====Electrophoresis====
-
<p>
+
[[image:HokkaidoU2012 120708 K346007 miniprep and pt7 rbs.jpg|thumb|plasmid extraction result (With ligation result of pT7+RBS+pSB1K3)]]
-
[[image:HokkaidoU2012 120708 K346007 miniprep and pt7 rbs.jpg|thumb|mini-prep result (With ligation result of pT7+RBS+pSB1K3)]]
+
Electrophoresis for plasmid extraction product(Ag43).
-
Erectrophoresis for mini-prep product(Ag43).
+
#Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.
-
#Prepared 1% Agalose gel and added EtBr then pre-migration in 30 min.
+
#1 ul 1kb ladder, 1.2 ul plasmid extraction product(1 ul is DNA solution and 0.2 ul is loading dye) added then migtrated.
-
#1 ul 1kb ladder, 1.2 ul mini-prep product(1 ul is DNA solution and 0.2 ul is loading dye) added then migtrated.
+
 
-
</p>
+
====Glycerol stock====
-
===Glycerol stock===
+
-
<p>
+
Made glycerol stock of K346007 (Ag43).
Made glycerol stock of K346007 (Ag43).
-
#Parts written above were cultivated in LBC.
+
#Parts written above were incubated in LBC.
#Added glycerol and freezed at -80C
#Added glycerol and freezed at -80C
-
</p>
 
*(Ag43 + dT)
*(Ag43 + dT)
Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)
Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)
-
===Digestion===
+
 
-
<p>
+
====Digestion====
Digested Ag43 and dT in solution by recipes Written below.
Digested Ag43 and dT in solution by recipes Written below.
-
'''Insert DNA required too much weight and volume'''(volume was calculated from concentration of DNA mini-prep
+
'''Insert DNA required too much weight and volume'''(volume was calculated from concentration of plasmid extraction
product)from our calculation. There are no insurance of succession of digestion.  
product)from our calculation. There are no insurance of succession of digestion.  
*Ag43(Insert)
*Ag43(Insert)
Line 310: Line 264:
|60 ul
|60 ul
|}
|}
 +
 +
*dT(Vector)
*dT(Vector)
3318bp(Ag43 + pSB1AK3)
3318bp(Ag43 + pSB1AK3)
Line 338: Line 294:
Digestion would be succeeded.
Digestion would be succeeded.
About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.
About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.
-
</p>
+
====Ethanol precipitation====
-
===Ethanol precipitation===
+
-
<p>
+
Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))
Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))
#Added 5 ul of NaoAc , 1.5 ul of glycogen and 125 ul of 100% ethanol to 50 ul DNA solutions.
#Added 5 ul of NaoAc , 1.5 ul of glycogen and 125 ul of 100% ethanol to 50 ul DNA solutions.
-
#Centrifuged in 15000 rpm, 10min at 4C.
+
#Centrifuged at 15000 rpm, 10min at 4C.
-
#Remove supernatant and added 220 ul of 70% ethanol.
+
#Removed supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000 rpm, 5 min at 4C.
+
#Centrifuged at 15000 rpm, 5 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 10 ul of DW.
+
#Removed supernatant and air drying at room temperature then added 10 ul of DW.
-
</p>
+
====Ligation====
-
===Ligation===
+
-
<p>
+
All DNA solutions were digested.
All DNA solutions were digested.
Used Ligation Mighty Mix(TakaraBio)
Used Ligation Mighty Mix(TakaraBio)
Line 384: Line 336:
|Hold
|Hold
|}
|}
-
</p>
+
====Electrophoresis====
-
===Electrophoresis===
+
-
<p>
+
[[image:HokkaidoU2012 120708 K346007-dT ligation.jpg|thumb|Erectrophoresis result]]
[[image:HokkaidoU2012 120708 K346007-dT ligation.jpg|thumb|Erectrophoresis result]]
Confirmation of succession of ligation.
Confirmation of succession of ligation.
-
#Prepared 1% Agalose gel and added EtBr then pre-migration in 30 min.
+
#Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.
#Added 1kb ladder, Ligation product(1 ul) and digestion products (control:each solutions 1 ul).
#Added 1kb ladder, Ligation product(1 ul) and digestion products (control:each solutions 1 ul).
-
#Migtrated in 30 min.
+
#Migtrated for 30 min.
-
</p>
+
====Transformation====
-
===Transformation===
+
-
<p>
+
Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.
Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.
-
#Stood on ice in 30 min.
+
#Incubated on ice for 30 min.
#Added 600 ul of LB to transformed DH5&alpha; solution.
#Added 600 ul of LB to transformed DH5&alpha; solution.
#From 700 solution(100 ul is DH5&alpha; and 600 ul is LB), 100 ul add to 900 ul of LB(x10 solution)
#From 700 solution(100 ul is DH5&alpha; and 600 ul is LB), 100 ul add to 900 ul of LB(x10 solution)
#Spread 300 ul from 600(700-100) ul and 1000 ul of LB&DH5&alpha; solution to each LBA plates.
#Spread 300 ul from 600(700-100) ul and 1000 ul of LB&DH5&alpha; solution to each LBA plates.
-
#Cultivated.
+
#Incubated for 19 hrs.
-
</p>
+
</div></div>
</div></div>

Latest revision as of 01:44, 27 September 2012

July 3rd

Get set...

July 4th

Transformation

Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) into DH5α

  1. Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice for 30 min.
  2. Plated them on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). ]

To gain chloramphenicol resistances transformed cell solution was incubated for 2 hrs. pT7 plate was incubated for 21 hrs and Others for 20 hrs.

July 5th

Transformation

K346007(Ag43) failed to grow on LBC plate. Transformation of K346007(Ag43) into DH5α.

  1. Mixed 1 ul DNA solution with DH5α competent cells and incubated on ice for 30 min.
  2. To gain chrolamphenicol resistance solution was pre-incubated for 2 hrs.
  3. Incubated on LBC for 21 hrs.

Single colony isolation

Performed single colony isolation of BBa_B0015, B0034, I179005 and K542009.

  1. Incubated the plates for 14 hrs and 30 mins

After consulting part page we found out that BBa_K542009 was Ag43 basic part, not composite! And the part didn't have Biobrick suffix.

July 6th

Incubation for plasmid extraction and glycerol stock

Incubated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) solution.

  1. Picked up two colonies from each plate.
  2. First colony was re-suspended in 1 ml LB (A and C respectively) for glycerol stock. Second colony was re-suspended in 2 ml LB(A and C respectively) for plasmid extraction.
  3. Incubated for 16 hrs.

Single colony isolation

  1. Single colony isolation of K346007(Ag43) on LBC.

July 7th

Incubation of plasmid extraction and glycerol stock

Incubation of (Ag43) in LBC liquid medium. Two colonies were re-suspended in 2 ml LBC. We incubated them at 38C.

One of the tubes was incubated for 8 hrs. It's for glycerol stock.

3A assembly!!!

Assembled pT7, RBS and pSB1C3 by 3A assembly. This is our first try!

Plasmid extraction

  1. Plasmid extraction of dT,RBS,pT7 and pLacI-RBS-Ag43. We used FastGene Plasmid Mini Kit(Nippon Gene)
  2. We got 50 ul of DNA solutions.

Glycerol stock

Glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.

  1. Parts written above were incubated in 1 ml of LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) for 16 hrs 30 min.
  2. Add glycerol and Freeze at -80C

Estimation of plasmid concentration

Erectrophoresis result

Electrophoresis to estimate the concentration of plasmid extraction products(dT,RBS,pT7 and pLacI-RBS-Ag43).

  1. Used 1% agarose gel.
  2. Added EtBr to TBE buffer and soaked the gel by applying current.
  3. Ran 1.2 ul of DNA solutions (1 ul is plasmid extraction product and 0.2 ul is Loading Dye) for 35 min.
  4. Took a photograph of 1% agarose gel.

Digestion of I719005, B0034 and pSB1K3

Digestion reaction

All parts were digested in 30 ul reaction solution.

  • I719005(40 ng/ul)
DNA solution 12.5 ul
EcoRI 1 ul
SpeI 1 ul
10xH Buffer 3 ul
DW 12.5 ul


  • B0034(40 ng/ul)
DNA solution 12.5 ul
XbaI 1 ul
PstI 1 ul
10xM Buffer 3 ul
DW 12.5 ul


  • pSB1K3(25 ng/ul)
DNA solution 12 ul
EcoRI 1 ul
PstI 1 ul
10xH Buffer 3 ul
DW 13 ul

Ethanol precipitation of Digestion products

Concentrating the DNA solution which and removing restriction enzymes.

  1. Added 3 ul of NaoAc, 1.5 ul of glycogen and 75 ul of 100% ethanol.
  2. Centrifuged at 14000 rpm, 30 min at 4C.
  3. Removed supernatant and added 220ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 15 min at 4C.
  5. Removed supernatant and air dried at room temperature. Dissolved in 10 ul of DW.

Ligation (3A Assembly)

3A assembly requires Ligation reaction total volume to be 25 ul.

Ligation Mighty Mix 12.5 ul
pT7 2 ul
RBS 2 ul
pSB1K3 2 ul
DW 6.5 ul
Total 25 ul


Incubation of ligation reaction.

Degree Minute
16 30
65 10
4 Hold


ligation was finished. But now is 10 pm. 2.5hrs are needed for transformation. Transformation would be finished at 0:30 am.

Withdraw!!!!

July 8th

  • (pT7 + RBS)

Transformation

Transformation for pT7+RBS+pSB1K3

  1. Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
  2. Stood on ice for 30 min.
  3. Added 600 ul of LB to transformed DH5α solution.
  4. Pre-incubated for 2 hrs at 37C.
  5. Splead 300 ul of LB&DH5α solution to LBK.
  6. Incubated for 19 hrs.

Plasmid extraction

Plasmid extraction for Liquid culture product of K346007(Ag43)

  1. Used FastGene Plasmid Mini Kit(Nippon Genetics)
  2. Elutioned in 50 ul
  3. First we eluted in collection tube. then moved in micro-centrifuge tube.

Electrophoresis

plasmid extraction result (With ligation result of pT7+RBS+pSB1K3)

Electrophoresis for plasmid extraction product(Ag43).

  1. Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.
  2. 1 ul 1kb ladder, 1.2 ul plasmid extraction product(1 ul is DNA solution and 0.2 ul is loading dye) added then migtrated.

Glycerol stock

Made glycerol stock of K346007 (Ag43).

  1. Parts written above were incubated in LBC.
  2. Added glycerol and freezed at -80C
  • (Ag43 + dT)

Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)

Digestion

Digested Ag43 and dT in solution by recipes Written below. Insert DNA required too much weight and volume(volume was calculated from concentration of plasmid extraction product)from our calculation. There are no insurance of succession of digestion.

  • Ag43(Insert)

5190bp(Ag43 + pSB1C3)

DW
DNA solution 48 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 6 ul
4 ul
Total 60 ul


  • dT(Vector)

3318bp(Ag43 + pSB1AK3)

DNA solution 8 ul
EcoRI 1 ul
XbaI 1 ul
10xM buffer 2 ul
DW 8 ul
Total 20 ul
Digestion result

K346007(Ag43) was 5190bp before digestion (Biobrick prefix + Ag43 + Biobrick suffix + pSB1C3). After digestion, Ag43 and pSB1C3 was separated and became fragments about 3120bp(Ag43) and 2070bp(pSB1C3). Gel image above shows there are two fragments, one fragment is about 2000bp other fragment is 3000bp. Digestion would be succeeded. About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.

Ethanol precipitation

Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))

  1. Added 5 ul of NaoAc , 1.5 ul of glycogen and 125 ul of 100% ethanol to 50 ul DNA solutions.
  2. Centrifuged at 15000 rpm, 10min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 5 min at 4C.
  5. Removed supernatant and air drying at room temperature then added 10 ul of DW.

Ligation

All DNA solutions were digested. Used Ligation Mighty Mix(TakaraBio)

Ligation Mighty Mix 5 ul
Insert: Ag43 2 ul
Vector: dT 2 ul
DW 1 ul
Total 10 ul

Ligation reaction recipe is following.

Degree Minute
16 30
65 10
4 Hold

Electrophoresis

Erectrophoresis result

Confirmation of succession of ligation.

  1. Prepared 1% Agalose gel and added EtBr then pre-migration for 30 min.
  2. Added 1kb ladder, Ligation product(1 ul) and digestion products (control:each solutions 1 ul).
  3. Migtrated for 30 min.

Transformation

Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.

  1. Added DNA soltions (Ligation products) 1 ul to DH5α compitent cell.
  2. Incubated on ice for 30 min.
  3. Added 600 ul of LB to transformed DH5α solution.
  4. From 700 solution(100 ul is DH5α and 600 ul is LB), 100 ul add to 900 ul of LB(x10 solution)
  5. Spread 300 ul from 600(700-100) ul and 1000 ul of LB&DH5α solution to each LBA plates.
  6. Incubated for 19 hrs.