Difference between revisions of "Team:AUC TURKEY/Experiments"

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{{AUC_TURKEY}}
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<h2>Experiments &amp; Protocols</h2>
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= Protocols =
  
 +
== Procedures for LB Agar Preparation ==
  
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<html>
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* In a steril environment, the tare of the container should be measured and subtracted from the overall weight.
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<head>
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* 7 grams of LB Agar is put in the container.
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<!-- Styles -->
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* 200 ml distilled water or is put into a graduated cylinder.
 +
* These two are mixed in a beaker.
 +
* The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.
 +
* Autoclave tape is sticked on to the aliminium.
 +
* The beaker is placed in to the autoclave machine.
 +
* Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.
 +
* After closing the lid of the machine, the 90 minute autoclave process is given start.
 +
* Take out the beaker and add antibiotics if required.
  
 +
== Warnings for the Autoclave! ==
  
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<h1>Protocols</h1>
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* Use only demineralised or disttiled water with the device.
 +
* Do not open the cover until the manometer drops to zero during the operation.
 +
* Please do not use the autoclave for other purposes than sterilization and agar.
 +
* Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.
 +
* Please be cautious when you are closing the lid not to trap your hand.
 +
* Please beware of the steam exhaust when you are opening to autoclave after sterilization.
 +
* Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.
  
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<p>.<p>
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== Procedures for LB Broth Preparation ==
  
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<div class="technology">1. Colony PCR</div>
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* In a steril environment, the tare of the container should be measured and subtracted from the overall weight.
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<div class="thelanguage">
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* 7 grams of LB Broth is put in the container.
 +
* 200 ml distilled water or is put into a graduated cylinder.
 +
* These two are mixed in a beaker.
 +
* The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.
 +
* Autoclave tape is sticked on to the aliminium.
 +
* The beaker is placed in to the autoclave machine.
 +
* Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.
 +
* After closing the lid of the machine, the 90 minute autoclave process is given start.
 +
* Take out the beaker and add antibiotics if required.
  
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<ol>
+
== Warnings for the Autoclave! ==
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<li>Pick a single colony into 5ul of NFW. (Fresh colonies grown that day work best, but they can also come from 4 C).</li>
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−
<li>Boil for 5 min at 95C.</li>
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−
</ol>
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−
<h3>PCR Reaction</h3>
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−
<p>Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4 C.</p>
+
  
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<img src="Buraya bir resim yerleştirin.">
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* Use only demineralised or disttiled water with the device.
 +
* Do not open the cover until the manometer drops to zero during the operation.
 +
* Please do not use the autoclave for other purposes than sterilization and agar.
 +
* Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.
 +
* Please be cautious when you are closing the lid not to trap your hand.
 +
* Please beware of the steam exhaust when you are opening to autoclave after sterilization.
 +
* Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.
  
 +
== Procedures for Transformation ==
  
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<div class="technology">2. Compotent Cell Preparation</div>
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* Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination.
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<div class="thelanguage">
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* Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation.
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<p><strong>For E.coli(DH-5&alpha;)</strong></p>
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* Take 10 ul plasmid and place them in 1.5 ml centrifuge tubes.
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</br>
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* Add 100 ul competent cells to the plasmid.
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<p>1-) Streak DH5&alpha; directly from a frozen stock onto LB agar plate (Amp-).</p>
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* Incubate the cells in ice for 20 minutes.
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<p>2-) Incubate at 37℃ for 12 h.</p>
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* After 20 minutes, heat the tubes in the 37 C heat block for 60 seconds.
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<p>3-) Inoculate one well isolated colony into 5ml of LB(Amp-).</p>
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* The same tubes should be placed in ice and should be incubated for 3 minutes.
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<p>4-) Incubate at 37℃ until OD<sub>600</sub> =0.8</p>
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* Afterwards, 900 ul LB should be added to the cells to complete them to 1000 ul.
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<p>5-) Transfer the preculture 5ml to 250ml of LB(Amp-)</p>
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* The microcentrifuge tubes are then sticked to the shaker horizantally and shaked for 1 hour with 320 rpm at 37 C.
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<p>6-) Incubate at 23℃ until OD<sub>600</sub> =0.4-0.5 (120rpm/min )</p>
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* 150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread.
−
<p>7-) Place sample on ice for 10min</p>
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* It is then spread on the plate and the plates are incubated at 37 C for 16 hours.
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<p>8-) Transfer the culture to steril ice cold tube(250ml)</p>
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<p>9-) 3.5Krpm at 4℃ for 5min</p>
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<p>10-) Remove the sup well</p>
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<p>11-) Resuspend the pellet by gently vortexing and pippeting in 100 ml of ice cold TB*</p>
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<p>12-) Sit on ice for 10min</p>
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<p>13-) 3K rpm at 4℃for 5min</p>
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<p>14-) Remove the sup well</p>
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<p>15-) Resuspend the pellet gently in 25ml of TB</p>
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<p>16-) Add 875&mu;l DMSO with disposable pippet</p>
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<p>17-) Mix gently by swirling</p>
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<p>18-) Sit on ice for 10min</p>
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<p>19-) Add 875&mu;l DMSO</p>
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<p>20-) Mix gently by swirling</p>
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<p>21-) Sit on ice for a few minuites</p>
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<p>22-) Dispense the sample into 1.5ml tube sterilized with UV for 10min</p>
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<p>23-) Chilled in liquid N2</p>
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<p><span style="font-size: 1.17em;"><br /></span></p>
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<p><span style="font-size: 1.17em;">[Preparation TB]</span></p>
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<br/>
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<p>1-) Add the following into 475 ml milli-Q</p>
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<p>2-) PIPES 1.5g</p>
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<p>3-) CaCl2･2H2O 1.1g</p>
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<p>4-) KCl 9.3g</p>
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<p>5-) Adjust pH to 6.7 with 1N KOH</p>
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<p>6-) Add 5.45g of MnCl2･4H2O</p>
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<p>7-) Add milli-Q upto 500ml</p>
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<p>8-) Filtration</p>
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 +
== Procedures for Isolation ==
  
 +
* The falcons are then centrifuged at 13,000 rpm for 5 minutes at room temperature.
 +
* After the centrifuge, the supernatent should be disposed without taking any pellets along with it.
 +
* The pelleted cells should be suspended in 250 ul Resuspension Solution and the tubes should be vortexed so that no cell clumps remain.
 +
* 250 ul Lysis Solution is added. The tubes are inverted 4-6 times but the inversion shouldn't be done really fast as the Lysis Solution must not be shaken. Also it is smart to heat the Lysis Solution in order to ensure that no pericipitate remain.
 +
* 350 ul Neutralization Solution should be added and the tube should be inverted immediately and throughly by inverting 4-6 times.
 +
* Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.
 +
* Transfer the supernatent to a spin column without taking any of the pellets.
 +
* Centrifuge the spin column for 1 minutes and discard the liquid at the bottom. Place the column at the same tube again.
 +
* Add 500 ul Wash Solution and centrifuge for 30-60 seconds. Discard the flow-through and place column back in.
 +
* Repeat the same process again with 500 ul Wash Solution.
 +
* Centrifuge for an additional 1 minute.
 +
* Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air.
 +
* Add 40 ul Nuclease Free Water which heated on 70 C  to the center of the silica membrane to elute the plasmid DNA. The pipette tip shouldn't the membrane. Incubate for 2 minutes and centrifuge for 3 minutes afterwards.
 +
* Discard the spin column and store at -20 C.
  
 +
== Digestion Protocol ==
  
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<div class="technology">3. Gel Electrophorosesis</div>
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* Take the average of the nucleic acid concentrations measured by the spectrometer.
−
<div class="thelanguage">
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* Divide 500 by the DNA average..
 +
* Add 1 ul of the enzymes with barrier tips.
 +
* Add 2 ul of the buffers
 +
* Subtract the amount of DNA from 16 ul. This result will be the amount of NFW used.
 +
* Add the NFW with barrier tips and do one pippetting while taking the NFW.
 +
* Then the DNA is put into the place which heat is 37 C and is left there for  1 or 3 hours.
  
 +
== Ligation Protocol ==
  
−
<p><strong>Standard %0.8 Agarose Gel Preparation</strong></p>
+
* 100ng vector is put into a eppendorf.
−
<ol>
+
* 2000ng plasmid is also added..
−
<li>Measure out 0.8 g of agarose</li>
+
* 2ul T4 Dna ligase Buffer is inserted to the mixture.
−
<li>Pour agarose powder into a microwavable flask along with 100mL of 1xTBE</li>
+
* 1ul T4 DNA ligase is then added with barrier tips.
−
<li>Microwave for 3 mins (until the agarose has dissolved completely and there is a nice rolling boil).</li>
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* Subtract the amount of DNA from 17 ul. This result will be the amount of NFW used.
−
<li>Let agarose solution cool down for 5min.</li>
+
* Then the DNA is hold in room tempature for 2 hours
−
<li>Add 3.6 ul EtBr and pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).</li>
+
−
</ol>
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−
<p>Wait 20-40 mins until poured gel has completely solidified.</p>
+
  
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<p style="text-align:right;font-size:1.3em;"><a href="" class="collapseLink" onClick="ddaccordion.collapseone('technology', 5); return false">[Collapse]</a></p>
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== Gel Preparation ==
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</div>
+
  
 +
* Mix 100ml TAE and 1 gram agarose in a glass beaker.
 +
* The mixture is then heated in a microwave for 3 minutes.
 +
* Let agarose solution cool down for 5min.
 +
* Afterwards, 4 ul EtBr is added and the beaker is mixed on a magnetic mixer.
 +
* Mold them and wait for 20 minutes fort he gel to harden.
  
 +
== Electrophoresis Protocol ==
  
−
<div class="technology">4.Gel Purification</div>
+
* Put 3 ul coloring agent on a strand of parafilm.
−
<div class="thelanguage">
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* Take 7 ul plasmid and do pipeting with the colouring agent.
−
 
+
* Switch the pipette to 10 ul and take the colored plasmid.
−
<p><strong>Gel Purification</strong></p>
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* Place the plasmid into one of the holes of our gel.
−
<p><strong></strong><strong>Gel purification using&<em>Thermo Scientific GeneJET Gel Extraction Kit</em></strong></p>
+
* Give electricity to the anode and cathode in required amounts.
−
<p>-All purification steps should be carried out at <strong>room temperature</strong>.</p>
+
* Wait according to the tank and the amount of electricity.
−
<p>-All centrifugations should be carried out in a table-top microcentrifuge at <strong>sup12000 x g</strong></p>
+
* Use the UV camera to get the results.
−
<p><strong></strong>Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.</p>
+
−
<ol>
+
−
<li><strong><em>Note</em></strong>. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.</li>
+
−
<li>Add <strong>1:1 volume</strong> of <strong>Binding Buffer</strong> to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).<br /> <strong><em>Note</em></strong>. For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.</li>
+
−
<li>Incubate the gel mixture at <strong>50-60&deg;C</strong> for <strong>10 min</strong> or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.</li>
+
−
<li><em>Optional</em>: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly.</li>
+
−
<li>Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br /> <strong><em>Note</em></strong>. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.</li>
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−
<li><em>Optional</em>: use this additional binding step only if the purified DNA will be used for sequencing. Add <strong>100 ul</strong> of <strong>Binding Buffer</strong> to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.</li>
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−
<li>Add 700 ul of <strong>Wash Buffer</strong> (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.</li>
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−
<li>Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.<br /> <strong><em>Note</em></strong>. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.</li>
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−
<li>Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add <strong>50 ul</strong> of <strong>NFW</strong> to the center of the purification column membrane. Centrifuge for 1 min.<br /> <strong><em>Note</em></strong>. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65&deg;C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.</li>
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−
<li>Discard the GeneJET purification column and store the purified DNA at -20&deg;C.</li>
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</ol>
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 6); return false">[Collapse]</a></p>
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</div>
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<div class="technology">5. LB Agar Preparation</div>
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<div class="thelanguage">
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<p>1-) Add 200 mL of dH2O to a graduated cyclindar.</p>
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<p>2-) Transfer dH2O into glass bottle.</p>
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−
<p>3-) Add 7 gr of LB-agar powder</p>
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<p>4-) Autoclave the bottle.</p>
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−
<p>5-) After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)</p>
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<p>6-) Pour the plates .</p>
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 7); return false">[Collapse]</a></p>
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</div>
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<div class="technology">6. LB Broth Preparation</div>
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<div class="thelanguage">
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−
 
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−
<p>1-) Add 200 mL of dH2O to a graduated cyclindar.</p>
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<p>2-) Transfer dH2O into glass bottle.</p>
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−
<p>3-) Add 4 gr of LB powder</p>
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<p>4-) Autoclave the bottle.</p>
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<p>5-) After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)</p>
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 8); return false">[Collapse]</a></p>
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</div>
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<div class="technology">7. Midiprep</div>
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<div class="thelanguage">
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<p>Midiprep Using <strong>invitrogen</strong> Midiprep Kit</p>
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<p><strong>Preparing Cell Lysate</strong></p>
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<p>1. For high copy number plasmids, use 15&ndash;25 mL of an overnight LB culture per sample in a disposable 50-mL conical tube.</p>
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<p><strong>Note:</strong> If you are using &gt;25 mL of culture volume of high copy plasmids, add twice the amount of Resuspension Buffer (R3) with RNase A, Lysis Buffer (L7), and Precipitation Buffer (N3) as directed in steps 3, 4, and 5, below, for best results.</p>
+
−
<p>For low copy number plasmids, use 25&ndash;100 mL of an overnight LB culture per sample in a 50-mL tube.</p>
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−
<p>2. Harvest the cells by centrifuging the overnight LB culture at 4000 &times; g for 10 minutes. Remove all medium.</p>
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−
<p>3. Add 4 mL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the cells until homogeneous.</p>
+
−
<p>4. Add 4 mL Lysis Buffer (L7). Mix gently by inverting the capped tube until the lysate mixture is thoroughly homogenous. Do not vortex. Incubate at room temperature for 5 minutes.</p>
+
−
<p><strong>Note:</strong> Do not allow lysis to proceed for more than 5 minutes.</p>
+
−
<p>5. Add 4 mL Precipitation Buffer (N3) and mix immediately by inverting the capped tube until the mixture is thoroughly homogeneous. Do not vortex.</p>
+
−
<p>6. Centrifuge the mixture at &gt;12,000 &times; g for 10 minutes at room temperature.</p>
+
−
<p><strong>Note:</strong> If the pellet does not adhere to the bottom of the tube, incubate the tube at room temperature for 5 minutes to allow the lysate and gelatinous pellet to separate. Pipet the clear lysate into another, sterile tube and centrifuge at &gt;12,000 &times; g at room temperature for 5 minutes to remove any remaining cellular debris.</p>
+
−
<p>7. Proceed to Binding and Washing DNA, next page.</p>
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−
<p><strong>Binding and Washing DNA</strong></p>
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−
<p>1. Load the supernatant from step 6 (Preparing Cell Lysate) onto the equilibrated column. Allow the solution in the column to drain by gravity flow.</p>
+
−
<p>2. Wash the column twice with 10 mL Wash Buffer (W8). Allow the solution in the column to drain by gravity flow after each wash. Discard the flow-through.</p>
+
−
<p>3. Proceed to Eluting and Precipitating DNA. For DNA precipitation, you can use the PureLink&reg; HiPure Precipitator Module (page 34) which allows you to precipitate DNA within 10 minutes without using a centrifuge. Alternatively, follow Eluting and Precipitating DNA on page 14 to perform traditional DNA precipitation using centrifugation. Refer to the manual supplied with the PureLink&reg; HiPure Precipitator Module for a detailed protocol.</p>
+
−
<p><strong>Eluting and Precipitating DNA</strong></p>
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−
<p>1. Place a sterile 15-mL centrifuge tube (elution tube) under the column.</p>
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−
<p>2. Add 5 mL Elution Buffer (E4) to the column to elute the DNA. Allow the solution to drain by gravity flow. Do not force out any remaining solution. The elution tube contains the purified DNA. Discard the column.</p>
+
−
<p>3. Add 3.5 mL isopropanol to the elution tube. Mix well.</p>
+
−
<p><strong>Note:</strong> Proceed to the protocol described in the PureLink&reg; HiPure Precipitator manual after this step, if you are using the precipitator module.</p>
+
−
<p>4. Centrifuge the tube at &gt;12,000 &times; g for 30 minutes at 4&deg;C. Carefully remove and discard the supernatant.</p>
+
−
<p>5. Resuspend the pellet in 3 mL 70% ethanol.</p>
+
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<p>6. Centrifuge the tube at &gt;12,000 &times; g for 5 minutes at 4&deg;C. Carefully remove and discard the supernatant.</p>
+
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<p>7. Air-dry the pellet for 10 minutes.</p>
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<p>8. Resuspend the DNA pellet in 200 &micro;L TE Buffer (TE). For low copy number plasmids, use 100 &micro;L of TE Buffer.</p>
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<p><strong>Note:</strong> Occasionally, insoluble particles may be present. These particles do not influence the quality of the DNA and can be easily removed. To remove insoluble particles, centrifuge the DNA solution at high speed for 1 minute at room temperature. Transfer the supernatant (DNA sample) into a fresh tube. Storing DNA To avoid repeated freezing and thawing of DNA, store the purified DNA at 4&deg;C for immediate use or aliquot the DNA and store at &ndash;20&deg;C for long-term storage.</p>
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 9); return false">[Collapse]</a></p>
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</div>
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<div class="technology">8. Miniprep</div>
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<div class="thelanguage">
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<p><strong>Miniprep</strong></p>
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<p><strong></strong><strong>Miniprep using&nbsp;<em>Thermo Scientific GeneJET Plasmid Miniprep Kit</em></strong></p>
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<p>-All purification steps should be carried out at <strong>room temperature.</strong></p>
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<p>-All centrifugations should be carried out in a table-top microcentrifuge at <strong>sup12000 x g</strong></p>
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<ol>
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−
<li>Resuspend the pelleted cells in <strong>250 ul of the Resuspension Solution</strong>. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br /> <strong><em>Note</em></strong>. Ensure RNase A has been added to the Resuspension Solution.</li>
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−
<li>Add <strong>250 ul of the Lysis Solution </strong>and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.<br /> <strong><em>Note</em></strong>. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.</li>
+
−
<li>Add <strong>350 ul of the Neutralization Solution</strong> and mix immediately and thoroughly by inverting the tube 4-6 times.<br /> <strong><em>Note</em></strong>. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.</li>
+
−
<li>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.</li>
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<li>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.</li>
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<li>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br /> <strong><em>Note</em></strong>.Do not add bleach to the flow-through.</li>
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−
<li>Add <strong>500 ul of the Wash Solution</strong> (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.</li>
+
−
<li>Repeat the wash procedure (step 7) using <strong>500 ul of the Wash Solution</strong>.</li>
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−
<li>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.</li>
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<li>Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add <strong>50 ul of the NFW</strong> to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.&nbsp;<strong><em>Note</em></strong>. An additional elution step (<em>optional</em>) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70&deg;C before applying to silica membrane.</li>
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<li>Discard the column and store the purified plasmid DNA at -20&deg;C.</li>
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<div class="technology">10.Transformation</div>
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<p>1-) Aseptic conditions prepared (70% EtOH, Bunsen burner etc.)</p>
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<p>2-) Place 500 uL LB in epp into heat block(42˚C).</p>
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<p>3-)Thaw 50 uL competent cells on ice.</p>
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<p>4-)Add 2 uL plasmid into the competent cell epp and incubate for 5 min on ice .</p>
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<p>5-)Incubate at 42˚C for 30 sec in heat block. &nbsp;</p>
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<p>6-)Incubate for 2,5 &nbsp;min on ice.</p>
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<p>7-) Complete to 200 uL with pre-heated LB (42˚C).</p>
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<p>8-) Epp s adhered with tape to horizontal on shaker.</p>
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<p>9-)Incubate at 37 C for 30 min at 240 rpm.</p>
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<p>10-)Spread 125-150 uL from each tube on agar plates with suitable antibiotic.</p>
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<p>11-)Incubate plates at 37˚C 16 h .</p>
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Revision as of 19:24, 18 September 2015

37 °C

Protocols

Procedures for LB Agar Preparation

  • In a steril environment, the tare of the container should be measured and subtracted from the overall weight.
  • 7 grams of LB Agar is put in the container.
  • 200 ml distilled water or is put into a graduated cylinder.
  • These two are mixed in a beaker.
  • The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.
  • Autoclave tape is sticked on to the aliminium.
  • The beaker is placed in to the autoclave machine.
  • Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.
  • After closing the lid of the machine, the 90 minute autoclave process is given start.
  • Take out the beaker and add antibiotics if required.

Warnings for the Autoclave!

  • Use only demineralised or disttiled water with the device.
  • Do not open the cover until the manometer drops to zero during the operation.
  • Please do not use the autoclave for other purposes than sterilization and agar.
  • Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.
  • Please be cautious when you are closing the lid not to trap your hand.
  • Please beware of the steam exhaust when you are opening to autoclave after sterilization.
  • Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.

Procedures for LB Broth Preparation

  • In a steril environment, the tare of the container should be measured and subtracted from the overall weight.
  • 7 grams of LB Broth is put in the container.
  • 200 ml distilled water or is put into a graduated cylinder.
  • These two are mixed in a beaker.
  • The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.
  • Autoclave tape is sticked on to the aliminium.
  • The beaker is placed in to the autoclave machine.
  • Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.
  • After closing the lid of the machine, the 90 minute autoclave process is given start.
  • Take out the beaker and add antibiotics if required.

Warnings for the Autoclave!

  • Use only demineralised or disttiled water with the device.
  • Do not open the cover until the manometer drops to zero during the operation.
  • Please do not use the autoclave for other purposes than sterilization and agar.
  • Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.
  • Please be cautious when you are closing the lid not to trap your hand.
  • Please beware of the steam exhaust when you are opening to autoclave after sterilization.
  • Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.

Procedures for Transformation

  • Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination.
  • Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation.
  • Take 10 ul plasmid and place them in 1.5 ml centrifuge tubes.
  • Add 100 ul competent cells to the plasmid.
  • Incubate the cells in ice for 20 minutes.
  • After 20 minutes, heat the tubes in the 37 C heat block for 60 seconds.
  • The same tubes should be placed in ice and should be incubated for 3 minutes.
  • Afterwards, 900 ul LB should be added to the cells to complete them to 1000 ul.
  • The microcentrifuge tubes are then sticked to the shaker horizantally and shaked for 1 hour with 320 rpm at 37 C.
  • 150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread.
  • It is then spread on the plate and the plates are incubated at 37 C for 16 hours.

Procedures for Isolation

  • The falcons are then centrifuged at 13,000 rpm for 5 minutes at room temperature.
  • After the centrifuge, the supernatent should be disposed without taking any pellets along with it.
  • The pelleted cells should be suspended in 250 ul Resuspension Solution and the tubes should be vortexed so that no cell clumps remain.
  • 250 ul Lysis Solution is added. The tubes are inverted 4-6 times but the inversion shouldn't be done really fast as the Lysis Solution must not be shaken. Also it is smart to heat the Lysis Solution in order to ensure that no pericipitate remain.
  • 350 ul Neutralization Solution should be added and the tube should be inverted immediately and throughly by inverting 4-6 times.
  • Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.
  • Transfer the supernatent to a spin column without taking any of the pellets.
  • Centrifuge the spin column for 1 minutes and discard the liquid at the bottom. Place the column at the same tube again.
  • Add 500 ul Wash Solution and centrifuge for 30-60 seconds. Discard the flow-through and place column back in.
  • Repeat the same process again with 500 ul Wash Solution.
  • Centrifuge for an additional 1 minute.
  • Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air.
  • Add 40 ul Nuclease Free Water which heated on 70 C to the center of the silica membrane to elute the plasmid DNA. The pipette tip shouldn't the membrane. Incubate for 2 minutes and centrifuge for 3 minutes afterwards.
  • Discard the spin column and store at -20 C.

Digestion Protocol

  • Take the average of the nucleic acid concentrations measured by the spectrometer.
  • Divide 500 by the DNA average..
  • Add 1 ul of the enzymes with barrier tips.
  • Add 2 ul of the buffers
  • Subtract the amount of DNA from 16 ul. This result will be the amount of NFW used.
  • Add the NFW with barrier tips and do one pippetting while taking the NFW.
  • Then the DNA is put into the place which heat is 37 C and is left there for 1 or 3 hours.

Ligation Protocol

  • 100ng vector is put into a eppendorf.
  • 2000ng plasmid is also added..
  • 2ul T4 Dna ligase Buffer is inserted to the mixture.
  • 1ul T4 DNA ligase is then added with barrier tips.
  • Subtract the amount of DNA from 17 ul. This result will be the amount of NFW used.
  • Then the DNA is hold in room tempature for 2 hours

Gel Preparation

  • Mix 100ml TAE and 1 gram agarose in a glass beaker.
  • The mixture is then heated in a microwave for 3 minutes.
  • Let agarose solution cool down for 5min.
  • Afterwards, 4 ul EtBr is added and the beaker is mixed on a magnetic mixer.
  • Mold them and wait for 20 minutes fort he gel to harden.

Electrophoresis Protocol

  • Put 3 ul coloring agent on a strand of parafilm.
  • Take 7 ul plasmid and do pipeting with the colouring agent.
  • Switch the pipette to 10 ul and take the colored plasmid.
  • Place the plasmid into one of the holes of our gel.
  • Give electricity to the anode and cathode in required amounts.
  • Wait according to the tank and the amount of electricity.
  • Use the UV camera to get the results.