Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Basic Part"

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	<div class="ContentHolder">		<div class="ContentHolder">
−	<div class="Text1" id="First1"><Red>Preparation of Competent Cells (<i>E.coli</i>)</Red></div>
−	<div class="Text2">
−	<Green>Materials and Reagents :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li><i>Escherichia coli</i> DH5α</li>
−	<li>LB broth</li>
−	<li>TB buffer</li>
−	<li>DMSO</li>
−	<li>Liquid nitrogen</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Equipment :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Ice</li>
−	<li>Shaker</li>
−	<li>Spectrometer</li>
−	<li>Centrifuge</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Procedure :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part2">
−	<li>Inoculate 10μL <i>Escherichia coli</i> DH5α into 10mL LB broth (1:1000)</li>
−	<li>Grow for 12-16 hours at 37℃ with shaking</li>
−	<li>Inoculate 500μL <i>Escherichia coli</i> DH5α into 50mL LB broth (1:100)</li>
−	<li>Grow for 2 hours at 37℃ with shaking to O.D.<Sub>600</Sub>=0.4-0.6</li>
−	<li>Split the cell into two Falcon tubes, both contain 25 mL <i>Escherichia coli</i> DH5α</li>
−	<li>Centrifuge at 4℃, 3000rpm(800G) for 10 minutes</li>
−	<li>Discard the supernatant</li>
−	<li>Resuspend the cell pellet by gently adding 8.5mL TB buffer (1/3 V)</li>
−	<li>On ice for 10 minutes</li>
−	<li>Centrifuge at 4℃, 3000rpm(800G) for 10 minutes</li>
−	<li>Discard the supernatant using pipetman</li>
−	<li>Resuspend the cell pellet by gently adding 2mL TB buffer (1/12.5 V)</li>
−	<li>Add 150μL DMSO (7%)</li>
−	<li>On ice for 10 minutes</li>
−	<li>Transfer the cell to new eppendorfs with 60μL per tube</li>
−	<li>Freeze the cell in liquid nitrogen</li>
−	<li>Store the cell at -80℃</li>
−	</ol>
−	</div>
−	<div class="Text1" id="First2"><Red>DNA Dissolution</Red></div>
−	<div class="Text2">
−	<Green>Materials and Reagents :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>DNA (BioBricks/synthesized)</li>
−	<li>ddH<Sub>2</Sub>O</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Procedure :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part2">
−	<li>Add 10μL ddH<Sub>2</Sub>O</li>
−	<li>Wait for 1 minute until the DNA dissolve</li>
−	<li>Pipet several times and transfer the DNA to an eppendorf</li>
−	</ol>
−	</div>
−	<div class="Text1" id="First3"><Red>Agarose Gel Electrophoresis</Red></div>
−	<div class="Text2">
−	<Green>Materials and Reagents :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Agarose</li>
−	<li>1X TAE</li>
−	<li>Tracking dye</li>
−	<li>Marker (1kb/100bp)</li>
−	<li>EtBr</li>
−	<li>ddH<Sub>2</Sub>O</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Equipment :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Gel tray</li>
−	<li>Well comb</li>
−	<li>Electrophoresis tank</li>
−	<li>UV detector</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Procedure :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part2">
−	<div class="Text_TitleUnderline">Casting an Agarose Gel (m/v = 1.0% or 1.5%)</div>
−	<li>Measure out appropriate mass of agarose powder into a serum bottle</li>
−	<li>Add appropriate volume of 1X TAE</li>
−	<li>Let agarose solution cool down to acceptable temperature for bare hands</li>
−	<li>Pour the solution into a gel tray with the well comb in place, and push the bubbles away with a pipette tip</li>
−	<li>Let sit at room temperature for 30 minutes, until it has completely solidified</li>
−	<div class="Text_TitleUnderline">Loading Samples and Running the Gel</div>
−	<li>Place the gel as well as its tray into the electrophoresis tank containing 1X TAE  </li>
−	(Make sure that the surface is higher than the top of the gel and not overflow)
−	<li>Mix the samples with tracking dye (1/10 V) sufficiently</li>
−	<li>Load the samples into the each well</li>
−	<li>Load marker (usually in the first and last lane)</li>
−	<li>Set an appropriate voltage (full/half) and run the gel for 15-20 minutes</li>
−	<div class="Text_TitleUnderline">Imaging the Gel</div>
−	<li>Put the gel into a container filled with 1X TAE and EtBr, staining for 5 minutes</li>
−	<li>Replace EtBr solution with water and destain for 3 minutes</li>
−	<li>Put the gel in an UV detector and record the result</li>
−	</ol>
−	</div>
−	<div class="Text1" id="First4"><Red>Gel Extraction</Red></div>
−	<div class="Text2">
−	<Green>Materials and Reagents :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Gel/PCR buffer</li>
−	<li>W1 buffer</li>
−	<li>Wash buffer</li>
−	<li>ddH<Sub>2</Sub>O</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Equipment :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Cutter knife</li>
−	<li>Eppendorf</li>
−	<li>Vortex mixer</li>
−	<li>Dry bath incubator</li>
−	<li>DF Column & Collection tube</li>
−	<li>Mini Centrifuge</li>
−	<li>Microcentrifuge</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Procedure :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part2">
−	<div class="Text_TitleUnderline">Gel Dissociation</div>
−	<li>Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (<300μL)</li>
−	<li>Transfer the gel slice to an eppendorf</li>
−	<li>Add 500μL Gel/PCR buffer to the sample and mix by vortex</li>
−	<li>Incubate at 60℃ for 10-15 minutes to ensure the gel slice has been completely dissolved (invert the tube every 2-3 minutes)</li>
−	<li>Cool the dissolved sample mixture to room temperature</li>
−	<div class="Text_TitleUnderline">DNA Binding</div>
−	<li>Place the DF Column in a 2mL Collection tube</li>
−	<li>Transfer 800μL of the sample mixture to the DF Column</li>
−	<li>Discard the flow-through and place the DF Column back in the Collection tube</li>
−	(If the sample mixture is more than 800μL, repeat the DNA binding step)
−	<div class="Text_TitleUnderline">Wash</div>
−	<li>Add 400μL W1 buffer into the DF Column</li>
−	<li>Spin down for approximately 20 seconds</li>
−	<li>Discard the flow-through and place the DF Column back in the Collection tube</li>
−	<li>Add 600μL wash buffer (contains ethanol) into the DF Column</li>
−	<li>Let stand for 1 minute at room temperature</li>
−	<li>Spin down for approximately 30 seconds</li>
−	<li>Discard the flow-through and place the DF Column back in the Collection tube</li>
−	<li>Centrifuge at 12000 rpm for 3 minutes to dry the column matrix</li>
−	(Can be done twice)
−	<li>Discard the flow-through</li>
−	<div class="Text_TitleUnderline">DNA Elution</div>
−	<li>Transfer the dried DF Column to a new eppendorf (cap cut)</li>
−	<li>Add 30μL of 37℃ ddH2O into the center of the column matrix</li>
−	<li>Let stand for at least 2 minutes to ensure that ddH2O is completely absorbed</li>
−	<li>Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA</li>
−	<li>Re-add the subnatant into the center of the column matrix</li>
−	<li>Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA</li>
−
−	</ol>
−	</div>
	<div class="Text1" id="First5"><Red>DNA Ligation</Red></div>		<div class="Text1" id="First5"><Red>DNA Ligation</Red></div>
	<div class="Text2">		<div class="Text2">
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	</ol>		</ol>
	</div>		</div>
−
−	<div class="Text1" id="First6"><Red>Plasmid DNA Extraction Using Alkaline Lysis Method (<i>E. coli</i>)</Red></div>
−	<div class="Text2">
−	<Green>Materials and Reagents :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li><i>Escherichia coli</i> DH5α (with plasmid)</li>
−	<li>LB broth</li>
−	<li>Resuspension solution (MPI, with RNAase)</li>
−	<li>Lysis solution (MPII)</li>
−	<li>Neutralizing solution (MPIII)</li>
−	<li>Isopropanol</li>
−	<li>70% ethanol</li>
−	<li>ddH<Sub>2</Sub>O</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Equipment :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Shaker</li>
−	<li>Centrifuge</li>
−	<li>Vortex mixer</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Procedure :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part2">
−	<li>Grow bacteria in LB broth with appropriate antibiotics at 37℃ overnight with shaking</li>
−	<li>Transfer 1.5mL culture to an eppendorf</li>
−	<li>Centrifuge at 4℃, 5000rpm for 5 minutes</li>
−	<li>Discard the supernatant</li>
−	<li>Add 150μL of resuspension solution (MPI, with RNAase) into each tube, pipet several times, and vortex to completely resuspend the cell pellet</li>
−	<li>Add 150μL of lysis solution (MPII), gently invert the tubes about 20 times, and then let the sample mixture stand at room temperature for 2 minutes</li>
−	<li>Add 150μL of neutralizing solution (MPIII) and mix by inverting the tubes about 20 times. Bacterial chromosomal DNA and proteins can be seen as white precipitates</li>
−	<li>Centrifuge at 4℃, 15000rpm for 15 minutes</li>
−	<li>Carefully transfer 400μL of the supernatant to a new eppendorf</li>
−	(Step 8&9 can be done twice)
−	<li>Add 400μL (same volume as the supernatant) isopropanol, and shake up the tubes as vigorously as one can</li>
−	<li>Centrifuge at 4℃, 15000rpm for 15 minutes</li>
−	<li>Discard the supernatant</li>
−	<li>Add 200μL of 70% ethanol to wash out the salts</li>
−	<li>Centrifuge at 4℃, 15000rpm for 5 minutes</li>
−	<li>Discard the supernatant and remove the remains as much as possible with pipetman</li>
−	<li>Air dry for about 30 minutes</li>
−	<li>Resuspend the DNA pellet with 20μL ddH<Sub>2</Sub>O</li>
−	</ol>
−	</div>
−	<div class="Text1" id="First7"><Red>Transformation (<i>E. coli</i>)</Red></div>
−	<div class="Text2">
−	<Green>Materials and Reagents :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>Competent cell (<i>Escherichia coli</i> DH5α)</li>
−	<li>LB plate (Amp+/CP+)</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Equipment :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part1">
−	<li>-80℃ refrigerator</li>
−	<li>Ice</li>
−	<li>37℃ incubator</li>
−	<li>Super optimal broth (SOB) (37℃)</li>
−	<li>Shaker</li>
−	</ol>
−	</div>
−	<div class="Text2">
−	<Green>Procedure :</Green>
−	</div>
−	<div class="Text3">
−	<ol class="part2">
−	<li>Take out the competent cells from -80℃ refrigerator</li>
−	<li>Thaw the cells on ice for 5 minutes</li>
−	<li>Add 1μL plasmid DNA into the competent cells, and mix gently by pipetting</li>
−	<li>On ice for 10 minutes</li>
−	<li>Heat shock at 37℃ for 3 minutes</li>
−	<li>On ice for 2 minutes</li>
−	<li>Add 150μL 37℃ SOB into the mixture</li>
−	<li>Place the tube at 37℃ with shaking for 1 hour(Amp+)/4 hours(CP+) respectively</li>
−	<li>Spread the cells onto the LB plates (Amp+/ CP+)</li>
−	<li>Incubate the plates at 37℃ with shaking for 12-16 hours</li>
−
−	</ol>
−	</div>
−
−
−
−
−
−
−
	</div>		</div>
	</div>		</div>

---

Revision as of 07:25, 7 September 2015

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  • Sub Item
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• Notes
  • Preparation of Competent Cells (E.coli)
  • DNA Dissolution
  • Agarose Gel Electrophoresis
  • Gel Extraction
  • DNA Ligation
  • Plasmid DNA Extraction Using Alkaline Lysis Method (E.coli)
  • Transformation (E.coli)

DNA Ligation

Materials and Reagents :

1. DNA (insert & vector)
2. Ligation high ver.2

Equipment :

1. Cooling dry bath incubator

Procedure :

1. Table

   Components	Volume (μL)
   Insert	x
   Vector	y
   Ligation High	1
   Total	7

           Molar ratio: insert/vector = 3/1
           x + y = 6
   
2. Gently mix the solution by pipetting up and down
3. Incubate
   1. at 16℃ for 2 hours, or
   2. at 37℃ for 1 hour, or
   3. at 4℃ overnight
4. Proceed with bacterial transformation

Maintained by the iGEM team NTU-LIHPAO-Taiwan    ©2015 NTU-LIHPAO-Taiwan