Team:Tokyo Metropolitan/Project/Fiber/Protocol

From 2010.igem.org

(Difference between revisions)
(→Protocol3:PCR)
(→Protocol3:PCR)
Line 27: Line 27:
　<li>bunsen burner
　<li>bunsen burner
　<li>flask
　<li>flask
-
　<li>plate
+
　<li>plate(SANPLATEC)
　<li>spreader
　<li>spreader
　<li>pipette
　<li>pipette
Line 53: Line 53:
  <li>1L of LB media
  <li>1L of LB media
   <ul>
   <ul>
-
   <li>LB agar:35g (Becton, Dickinson)
+
   <li>LB agar:35g  
   <li>Distilled water:~1L
   <li>Distilled water:~1L
</ul></li></ul>
</ul></li></ul>
(If you want to obtain colony with plasmid, add appropriate　anti-biotic) 　
(If you want to obtain colony with plasmid, add appropriate　anti-biotic) 　
   </ul>
   </ul>
-
<br>
+
<br><br>
<font size="3"><span style="text-decoration:underline">Equipment</font></span>
<font size="3"><span style="text-decoration:underline">Equipment</font></span>
  <ul>
  <ul>
Line 66: Line 66:
  <li>bunsen burner
  <li>bunsen burner
  <li>flask
  <li>flask
-
  <li>plate
+
  <li>plate(SANPLATEC)
  <li>inoculating loop
  <li>inoculating loop
  <li>pipette
  <li>pipette
Line 97: Line 97:
  <ul><li>Pho polymerase(Nippon gene)
  <ul><li>Pho polymerase(Nippon gene)
  <li>KOD polymerase
  <li>KOD polymerase
-
  <li>Taq polymerase<br/>
+
  <li>Taq polymerase
-
</ul>
+
</ul></ul>
-
<br/>
+
<br>
<font size="3"><span style="text-decoration:underline">Equipment</font></span>
<font size="3"><span style="text-decoration:underline">Equipment</font></span>
Line 108: Line 108:
  <li>pipette
  <li>pipette
  <li>pipette tip
  <li>pipette tip
-
  </ul>
+
  </ul></ul>
-
<br/>
+
<br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
<ol><li>Add and mix above materials to each PCR tubes on the ice</li>
<ol><li>Add and mix above materials to each PCR tubes on the ice</li>
-
<li>add templete DNA (or cells) into the PCR tubes</li>
+
<li>Add templete DNA (or cells) into the PCR tubes</li>
<li>Setting PCR tubes in the thermal cycler
<li>Setting PCR tubes in the thermal cycler
-
<li>cycle of PCR
+
<li>Cycle of PCR
<ul><li>initialization  
<ul><li>initialization  
<li>denaturation
<li>denaturation
Line 136: Line 136:
  <li>1% agarose gel
  <li>1% agarose gel
   <ul>
   <ul>
-
   <li>agarose S
+
   <li>agarose S (Nippon gene)
   <li>TAE buffer  
   <li>TAE buffer  
   <li>ethidium bromide  
   <li>ethidium bromide  
Line 142: Line 142:
  <li>1×TAE buffer
  <li>1×TAE buffer
  <li>10×Loading buffer  
  <li>10×Loading buffer  
-
  <li>template DNA(PCR production)
+
  <li>template DNA(PCR/digestion production)
  </ul>
  </ul>
<br>
<br>
Line 149: Line 149:
  <li>microwave
  <li>microwave
  <li>gel box
  <li>gel box
-
  <li>flask(500ml)
+
  <li>flask
  </ul>
  </ul>
<br>
<br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
  <ol>
  <ol>
-
  <li>Measure out agarose into a beaker with the 300ml of TAE buffer.Microwave until the agarose is fully melted(5minutes×3).
+
  <li>Measure out agarose into a beaker with TAE buffer and Microwave until the agarose is fully melted(5minutes×3).
  <li>Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.  
  <li>Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.  
  <li>While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak.  
  <li>While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak.  
  <li>Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.
  <li>Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.
  <li>If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.
  <li>If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.
-
  <li>set agarose gel and add TAE buffer in gel box.  
+
  <li>Set agarose gel and add TAE buffer in gel box.  
-
  <li>mix DNA and Loading buffer and then put in well them(marker sets another well).
+
  <li>Mix DNA and Loading buffer and then put in well them(marker sets another well).
-
  <li>load DNA at 100V for two third of entire(about 15minutes).
+
  <li>Load DNA at 100V for two third of entire(about 15minutes).
-
  <li>image the consequence of electrophoreses.
+
  <li>Image the consequence of electrophoreses.
  </ol>
  </ol>
<br>
<br>
Line 169: Line 169:
</html>
</html>
-
==Protocol5-1:DNA purification from agarose gel==
+
==Protocol5:DNA extraction from agarose gel==
<html>
<html>
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　<font size="3"><span style="text-decoration:underline">Material</font></span>
　<font size="3"><span style="text-decoration:underline">Material</font></span>
  <ul>
  <ul>
-
  <li>QIAGEN(gel extraction kit)
+
  <li>QIAGEN Gel extraction kit
   <ul>
   <ul>
-
   <li>QG buffer 300µl
+
   <li>DNA in agarose gel
-
   <li>PE buffer 700µl
+
   <li>QG buffer  
-
   <li>EB buffer 50µl
+
   <li>PE buffer  
-
   <li>tube for column
+
   <li>EB buffer
   </ul>
   </ul>
  </ul>
  </ul>
Line 188: Line 188:
  <li>centrifuge
  <li>centrifuge
  <li>heating plate
  <li>heating plate
 +
<li>tube with column
  <li>pipette
  <li>pipette
  <li>pipette tip
  <li>pipette tip
 +
  </ul>
  </ul>
<br>
<br>
　<font size="3"><span style="text-decoration:underline">Procedure</font></span>
　<font size="3"><span style="text-decoration:underline">Procedure</font></span>
  <ol>
  <ol>
-
  <li>cut gel of electrophoreses
+
  <li>Cut a band of DNA in agarose gel
-
  <li>add pieces of gel to tubes
+
  <li>Add pieces of gel to tubes
-
  <li>take 300µl QG buffer into tubes and dissolve at 50°C
+
  <li>Take QG buffer into tubes and dissolve at 50°C
-
  <li>add a solution of QG buffer and gel to tubes for column
+
  <li>Add a solution of QG buffer and gel to tubes for column
-
  <li>centrifuge 15000rpm/1min
+
  <li>Centrifuge 15000rpm/1min
-
  <li>throw “flow-thru” away and take 700µl PE buffer
+
  <li>Throw flow-through away and take PE buffer
-
  <li>centrifuge 15000rpm/1min
+
  <li>Centrifuge 15000rpm/1min
-
  <li>throw flow-thru away  
+
  <li>Throw flow-through away  
-
  <li>centrifuge 15000rpm/1min
+
  <li>Centrifuge 15000rpm/1min
-
  <li>change tube for column
+
  <li>Change tube for column
-
  <li>add 50µl of EB buffer (aim to center of tube)
+
  <li>Add EB buffer (aim to center of tube)
-
  <li>centrifuge 15000rpm/1min
+
  <li>Centrifuge 15000rpm/1min
  </ol>
  </ol>
</td></tr></table></center></html>
</td></tr></table></center></html>
-
==Protocol5-2:DNA Purification with silica gel ==
+
==Protocol6:DNA Purification with silica gel ==
<font size="3"><span style="text-decoration:underline">Material</span>
<font size="3"><span style="text-decoration:underline">Material</span>
<font size="2">
<font size="2">
Line 237: Line 239:
#Add TE buffer and mix with Vortex
#Add TE buffer and mix with Vortex
#Centrifuge 30sec
#Centrifuge 30sec
-
#DNA is solved in Supernatant
+
#Supernatant contains DNA
-
==Protocol6:Restriction enzyme digestion==
+
==Protocol7:Restriction enzyme digestion==
<html>
<html>
Line 245: Line 247:
　<font size="3"><span style="text-decoration:underline">Material</font></span>
　<font size="3"><span style="text-decoration:underline">Material</font></span>
  <ul>
  <ul>
-
  <li>DNA for digestion 50µl
+
  <li>DNA for digestion  
-
  <li>10×M buffer 5µl
+
  <li>10×M buffer(Nippon gene)
-
  <li>XbaI 1µl
+
  <li>XbaI(Nippon gene)
-
  <li>SpeI 1µl
+
  <li>SpeI(Nippon gene)
  </ul>
  </ul>
<br>
<br>
Line 254: Line 256:
  <ul>
  <ul>
  <li>incubater
  <li>incubater
-
  <li>PCR tube
+
  <li>tube
  <li>pipette
  <li>pipette
 +
<li>pipette tip
  <li>freezer
  <li>freezer
  <li>freezer box
  <li>freezer box
Line 262: Line 265:
　<font size="3"><span style="text-decoration:underline">Procedure</font></span>
　<font size="3"><span style="text-decoration:underline">Procedure</font></span>
  <ol>
  <ol>
-
  <li>add above materials to a tube
+
  <li>Add above materials to a tube
  <li>Incubate 37°C for 2~16hours
  <li>Incubate 37°C for 2~16hours
  </ol>
  </ol>
</td></tr></table></center></html>
</td></tr></table></center></html>
-
==Protocol7:Ligation==
+
==Protocol8:Ligation==
<html>
<html>
Line 290: Line 293:
　<font size="3"><span style="text-decoration:underline">Procedure</font></span>
　<font size="3"><span style="text-decoration:underline">Procedure</font></span>
  <ol>
  <ol>
-
  <li>Incubate at room temperature for 5minutes
+
<li>Add materials to 20μl
 +
  <li>Incubate at 16℃ for 5~30minutes
  </ol>
  </ol>
</td></tr></table></center></html>
</td></tr></table></center></html>
-
==Protocol8:Transformation ==
+
==Protocol9:Transformation ==
<html>
<html>
Line 300: Line 304:
<font size="3"><span style="text-decoration:underline">Material</font></span>
<font size="3"><span style="text-decoration:underline">Material</font></span>
  <ul>
  <ul>
-
  <li>E.coli Competent cell (JM109/NovaBlue)  
+
  <li>E.coli Competent cell (Ecos JM109(Nippon gene)/NovaBlue(Merck))
-
  <li>ligation production(refer to <a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80Ligation">Ligation</a>)  
+
  <li>DNA(for example ligation production)  
  </ul>
  </ul>
<br>
<br>
Line 310: Line 314:
  <li>heater
  <li>heater
  <li>bunsen burner
  <li>bunsen burner
-
  <li>flask(500ml)
+
  <li>plate(SANPLATEC)
-
<li>plate
+
  <li>tube
  <li>tube
  <li>pipette
  <li>pipette
Line 320: Line 323:
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
  <ol>
  <ol>
-
  <li>add 50µl of E.coli Competent cells to tubes which was used ligation
+
  <li>Mix 50µl of E.coli Competent cells and DNA
  <li>incubate the cells on ice for 30minutes
  <li>incubate the cells on ice for 30minutes
-
  <li>heat shock the cells by heater at42°C for 45sec  
+
  <li>heat shock the cells at 42°C for 45sec  
  <li>incubate the cells on ice for 2 min
  <li>incubate the cells on ice for 2 min
-
  <li>streak the cells on LB medium plate added chloramphenicol
+
<li>Add 4 times volume of SOC broth
 +
  <li>streak the cells on LB medium plate added anti-biotic
  <li>incubate cells at 37°C during for 14hours
  <li>incubate cells at 37°C during for 14hours
  </ol>
  </ol>
Line 331: Line 335:
</html>
</html>
-
==Protocol9:Miniprep (extraction of plasmid kit)==
+
==Protocol10:Miniprep (extraction of plasmid kit)==
<html>
<html>
Line 337: Line 341:
<font size="3"><span style="text-decoration:underline">Material</font></span>
<font size="3"><span style="text-decoration:underline">Material</font></span>
  <ul>
  <ul>
-
  <li>E.coli (plasmid)
+
  <li>Preculture of E.coli  
-
  <li>SolutionⅠ
+
<li>Bio-Rad extraction of plasmid kit
-
  <li>SolutionⅡ
+
  <ul><li>resuspention solution
-
  <li>SolutionⅢ
+
  <li>lysis Solution
-
  <li>matrix
+
  <li>neutralization　Solution
-
  <li>TE buffer
+
  <li>quantum prep mix
 +
  <li>wash buffer
 +
</ul><li>TE buffer
  </ul>
  </ul>
<br>
<br>
Line 352: Line 358:
  <li>tube
  <li>tube
  <li>filter
  <li>filter
 +
<li>pipette
 +
<li>pipette tip
  </ul>
  </ul>
<br>
<br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
  <ol>
  <ol>
-
  <li>pick E.coli cells up from a colony and add to 2ml tube
+
  <li>Add 2ml of preculture of E.coli into a tube
  <li>centrifuge 15000rpm/30sec and throw supernatant fluid away
  <li>centrifuge 15000rpm/30sec and throw supernatant fluid away
-
  <li>add 120µl of Solution I and vortex
+
  <li>add 120µl of resuspention solution and vortex
-
  <li>add 250µl of Solution II and shake with hand
+
  <li>add 250µl of lysis Solution and shake with hand
-
  <li>add 250µl of Solution III
+
  <li>add 250µl of neutralization　Solution and shake with hand
  <li>centrifuge 15000rpm/5min
  <li>centrifuge 15000rpm/5min
  <li>set spin filter in 2ml tube  
  <li>set spin filter in 2ml tube  
-
  <li>add supernatant to spin filter, then add 200µl of matrix and suspend with pipet
+
  <li>add supernatant to spin filter, then add 200µl of quantum prep mix and suspend with pipette
  <li>centrifuge 15000rpm/30sec and throw supernatant fluid away
  <li>centrifuge 15000rpm/30sec and throw supernatant fluid away
  <li>add wash buffer 500µl and then centrifuge 15000rpm/30sec and throw supernatant fluid away (twice)
  <li>add wash buffer 500µl and then centrifuge 15000rpm/30sec and throw supernatant fluid away (twice)
  <li>centrifuge 15000rpm/2min and throw supernatant fluid away
  <li>centrifuge 15000rpm/2min and throw supernatant fluid away
  <li>set spin filter in 1.5ml tube
  <li>set spin filter in 1.5ml tube
-
  <li>add TE buffer to 15ml falcon tube and heat 15min with microwave
+
  <li>add TE buffer to 15ml falcon tube and heat 15sec with microwave
  <li>add 100µl of TE buffer  
  <li>add 100µl of TE buffer  
  <li>centrifuge 15000rpm/1min
  <li>centrifuge 15000rpm/1min
  </ol>
  </ol>
<br>
<br>
 +
 +
==Protocol11:Sequence==
 +
<html>
 +
<center><table><tr><td width="850" align="left">
 +
<font size="3"><span style="text-decoration:underline">Material</font></span>
 +
<ul>
 +
<li>DNA
 +
<li>Big Dye
 +
<li>primer
 +
<li>DW
 +
<li>ethanol
 +
<li>EDTA
 +
<li>Hi-Di solution</ul></ul>
 +
<br/>
 +
 +
<font size="3"><span style="text-decoration:underline">Equipment</font></span>
 +
<ul>
 +
<li>sequencer
 +
<li>centrifuge
 +
<li>vortex
 +
<li>pipette
 +
<li>pipette tip</ul></ul>
 +
<br/>
 +
 +
<font size="3"><span style="text-decoration:underline">Procedure</font></span>
 +
<ol><li>mix DNA(<50ng),Big Dye,promer,ethanol and DW
 +
<li>PCR
 +
<li>Add EDTA and Ethanol and put at room temperature　(15min)
 +
<li>centrifuge (15000rpm,30min) and throw away supernatant
 +
<li>add ethanol again
 +
<li>centrifuge (15000rpm,15min) and throw away supernatant
 +
<li>add Hi-Di solution
 +
<li>heat at 95℃
 +
<li>transfer these sample to plate for sequence
 +
<li>read sequence
 +
<div>
<div>
</html>
</html>

Revision as of 20:45, 26 October 2010


E.coli Fiber Project Protocol

Spidertan.png


Contents

Protocol1:Grow up a culture of A.xylinum

Material 　
  • ''A.xylinum'' JCM 7664 strain 　
  • 1L of Acetobacter media(Open Wet Ware) 　　
    • Glucose:2.0g 　　
    • Peptone:5.0g 　　
    • Yeast extract:5.0g 　　
    • Na2HPO4:2.7g 　　
    • Citric acid:1.65g 　　
    • Distilled water:~1L
　(If you are making plates, use the same protocol but add 15g of agar.)

Equipment 　
  • autoclave 　
  • incubator 　
  • scale 　
  • bunsen burner 　
  • flask 　
  • plate(SANPLATEC) 　
  • spreader 　
  • pipette 　
  • pipette tip

Procedure 　
  1. Prepare media as outlined (add the materials as above) 　
  2. Autoclave to sterilize media(121°C　20minute). 　
  3. Streak/inoculate A.xylinum onto plates or in media. 　
  4. Incubate cells at 28°C for 2-3 days. 　

Note 　
  1. The growth of A.xylinum does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　
  2. The growth of A.xylinum is accompanied by the formation of a thick cellulose matrix within the media that must be removed before cells can be pelleted for a miniprep procedure. Simply vortex briefly to break up the cellulose into chunks and remove the cellulose chunks from the media with a pipette while carefully avoiding the removal of cells. 　
  3. A.xylinum will grow well at room temperature in aerobic conditions.

Protocol2:Grow up a culture of E.coli

Material
  • E.coli K12 strain
  • 1L of LB media
    • LB agar:35g
    • Distilled water:~1L
(If you want to obtain colony with plasmid, add appropriate　anti-biotic) 　

Equipment
  • autoclave
  • incubator
  • scale
  • bunsen burner
  • flask
  • plate(SANPLATEC)
  • inoculating loop
  • pipette
  • pipette tip

Procedure
  1. Prepare media as outlined (add the materials as above).
  2. Autoclave to sterilize media(121°C　20minute).
  3. Streak/inoculate E.coli onto plates or in media.
  4. Incubate cells at 37°C.

Protocol3:PCR

Material
  • forward Primer
  • reverse Primer
  • PCR buffer
  • dNTP mixture
  • milli-Q
  • DNA polymerase
    • Pho polymerase(Nippon gene)
    • KOD polymerase
    • Taq polymerase

Equipment
  • thermal cycler
  • vortex mixer
  • PCR-tubes
  • pipette
  • pipette tip

Procedure
  1. Add and mix above materials to each PCR tubes on the ice
  2. Add templete DNA (or cells) into the PCR tubes
  3. Setting PCR tubes in the thermal cycler
  4. Cycle of PCR
    • initialization
    • denaturation
    • annealing
    • elongation

      (denaturation～elongation　30cycles)

    • reaction stop

Protocol4:Agarose gel electrophoresis

Material
  • 1% agarose gel
    • agarose S (Nippon gene)
    • TAE buffer
    • ethidium bromide
  • 1×TAE buffer
  • 10×Loading buffer
  • template DNA(PCR/digestion production)

Equipment
  • microwave
  • gel box
  • flask

Procedure
  1. Measure out agarose into a beaker with TAE buffer and Microwave until the agarose is fully melted(5minutes×3).
  2. Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
  3. While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak.
  4. Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.
  5. If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.
  6. Set agarose gel and add TAE buffer in gel box.
  7. Mix DNA and Loading buffer and then put in well them(marker sets another well).
  8. Load DNA at 100V for two third of entire(about 15minutes).
  9. Image the consequence of electrophoreses.

Protocol5:DNA extraction from agarose gel

　Material
  • QIAGEN Gel extraction kit
    • DNA in agarose gel
    • QG buffer
    • PE buffer
    • EB buffer

　Equipment
  • centrifuge
  • heating plate
  • tube with column
  • pipette
  • pipette tip

　Procedure
  1. Cut a band of DNA in agarose gel
  2. Add pieces of gel to tubes
  3. Take QG buffer into tubes and dissolve at 50°C
  4. Add a solution of QG buffer and gel to tubes for column
  5. Centrifuge 15000rpm/1min
  6. Throw flow-through away and take PE buffer
  7. Centrifuge 15000rpm/1min
  8. Throw flow-through away
  9. Centrifuge 15000rpm/1min
  10. Change tube for column
  11. Add EB buffer (aim to center of tube)
  12. Centrifuge 15000rpm/1min

Protocol6:DNA Purification with silica gel

Material

  • Binding buffer
  • silica gel
  • wash buffer
  • TE buffer

Equipment

  • centrifuge
  • vortex
  • aspirator
  • pipette
  • pipette tip

Procedure

  1. Add 3 times Binding buffer than digestion production
  2. Add 10µl of silica gel and mix with Vortex
  3. Centrifuge 1min
  4. Remove supernatant with aspirator
  5. Add Wash buffer and mix with vortex
  6. Centrifuge 30sec
  7. Remove supernatant with aspirator
  8. Remove ethanol by drying
  9. Add TE buffer and mix with Vortex
  10. Centrifuge 30sec
  11. Supernatant contains DNA

Protocol7:Restriction enzyme digestion

　Material
  • DNA for digestion
  • 10×M buffer(Nippon gene)
  • XbaI(Nippon gene)
  • SpeI(Nippon gene)

　Equipment
  • incubater
  • tube
  • pipette
  • pipette tip
  • freezer
  • freezer box

　Procedure
  1. Add above materials to a tube
  2. Incubate 37°C for 2~16hours

Protocol8:Ligation

　Material
  • 2×ligation Mix(Nippon gene)
  • plasmid DNA
  • insert DNA

　Equipment
  • incubater
  • PCR tube
  • pipette
  • freezer
  • freezer box

　Procedure
  1. Add materials to 20μl
  2. Incubate at 16℃ for 5~30minutes

Protocol9:Transformation

Material
  • E.coli Competent cell (Ecos JM109(Nippon gene)/NovaBlue(Merck))
  • DNA(for example ligation production)

Equipment
  • autoclave
  • incubator
  • heater
  • bunsen burner
  • plate(SANPLATEC)
  • tube
  • pipette
  • pipette tip
  • ice

Procedure
  1. Mix 50µl of E.coli Competent cells and DNA
  2. incubate the cells on ice for 30minutes
  3. heat shock the cells at 42°C for 45sec
  4. incubate the cells on ice for 2 min
  5. Add 4 times volume of SOC broth
  6. streak the cells on LB medium plate added anti-biotic
  7. incubate cells at 37°C during for 14hours

Protocol10:Miniprep (extraction of plasmid kit)

Material
  • Preculture of E.coli
  • Bio-Rad extraction of plasmid kit
    • resuspention solution
    • lysis Solution
    • neutralization　Solution
    • quantum prep mix
    • wash buffer
  • TE buffer

Equipment
  • centrifuge
  • microwave
  • vortex mixer
  • tube
  • filter
  • pipette
  • pipette tip

Procedure
  1. Add 2ml of preculture of E.coli into a tube
  2. centrifuge 15000rpm/30sec and throw supernatant fluid away
  3. add 120µl of resuspention solution and vortex
  4. add 250µl of lysis Solution and shake with hand
  5. add 250µl of neutralization　Solution and shake with hand
  6. centrifuge 15000rpm/5min
  7. set spin filter in 2ml tube
  8. add supernatant to spin filter, then add 200µl of quantum prep mix and suspend with pipette
  9. centrifuge 15000rpm/30sec and throw supernatant fluid away
  10. add wash buffer 500µl and then centrifuge 15000rpm/30sec and throw supernatant fluid away (twice)
  11. centrifuge 15000rpm/2min and throw supernatant fluid away
  12. set spin filter in 1.5ml tube
  13. add TE buffer to 15ml falcon tube and heat 15sec with microwave
  14. add 100µl of TE buffer
  15. centrifuge 15000rpm/1min

==Protocol11:Sequence==
Material
  • DNA
  • Big Dye
  • primer
  • DW
  • ethanol
  • EDTA
  • Hi-Di solution

Equipment
  • sequencer
  • centrifuge
  • vortex
  • pipette
  • pipette tip

Procedure
  1. mix DNA(<50ng),Big Dye,promer,ethanol and DW
  2. PCR
  3. Add EDTA and Ethanol and put at room temperature　(15min)
  4. centrifuge (15000rpm,30min) and throw away supernatant
  5. add ethanol again
  6. centrifuge (15000rpm,15min) and throw away supernatant
  7. add Hi-Di solution
  8. heat at 95℃
  9. transfer these sample to plate for sequence
  10. read sequence