<i class="fa fa-file-text"></i><div class="text_font"> 2016.7.25</div>

<small class="text-navy"><div class="text_font">Morning</div></small>

                                     <div class="m-b-xs text_font"><strong>Making the agarose gel</strong></div>

<li><div class="text_font">Experiment reagent : agarose, TAE solution, SYBY GREEN</div></li>

<li><div class="text_font">Experiment apparatus:microwave oven, analytical balance, graduated cylinder100ml, conical flask500ml, plastic glove，the equipment for making gel(including the combs), electrophoresis tank</div></li>

<li><div class="text_font">Experiment steps:</div>

<li><div class="text_font">add agarose 0.8g, TAE solution 100μl into the conical flask </div></li>

<li><div class="text_font">put a plastic glove on the conical flask (prevent it from evaporating)</div></li>

<li><div class="text_font">put the conical flask into the microwave oven to heat up for 2min</div></li>

<li><div class="text_font">take the conical flask out, after it is cooled down, add nucleic acid dye SYBY GREEN 120μl into it , and then shake it until it is mixed uniform</div></li>

<li><div class="text_font">pour the solution into Agarose gel plate, until it is 4~6mm thick</div></li>

<li><div class="text_font">put an suitable comb in the plate, and then cool the gel down in indoor temperature</div></li>

<li><div class="text_font">after it is solidified completely, pull out the comb vertically to make sure the sample hole is all right</div></li>

<li><div class="text_font">put the gel into the electrophoresis tank, add TBE until it is over the gel 1~2mm</div></li>

                                     <i class="fa fa-file-text"></i><div class="text_font"> 2016.7.27</div>

<small class="text-navy"><div class="text_font">The whole day</div></small>

                                     <div class="m-b-xs text_font"><strong>PCR&AGE </strong></div>

                                         <li><div class="text_font">Experiment material:genome DNA of arabidopsis , primer（flu、cop1、plf1、phyB、pHhl1、hhl1）</div></li>

<li><div class="text_font">Experiment reagent:ddH2O, dNTP,Taq enzyme,Mg2+,10×Buffer</div></li>

<li><div class="text_font">Experiment apparatus:DNA thermal cycler, elctrophoresis apparatus,elctrophoresis tank,ultraviolet detector, table centrifuge, 0.5ml &1.5ml centrifuge tube, micropipet</div></li>

<li><div class="text_font">experiment steps:</div>

<li><div class="text_font">disposal the reaction system</div>

<li><div class="text_font">2μl buffer（including 15mM MgCl2）</div></li>

<li><div class="text_font">1μl dNTPs</div></li>

<li><div class="text_font">1.5μl DNA</div></li>

<li><div class="text_font">1.0μl forward primer</div></li>

<li><div class="text_font">1.0μl reverse primer</div></li>

<li><div class="text_font">0.2μl Taq enzyme</div></li>

<li><div class="text_font">13μl ddH2O</div></li>

<li><div class="text_font">(primer：flu,cop1,plf1,phyB,pHhl1,hhl1)</div></li>

<li><div class="text_font">Set the PCR</div>

<li><div class="text_font">94℃×5min + （94℃×30s +62℃×35s + 72℃×40s）×40+ 72℃×5min + 4℃</div></li>

<li><div class="text_font">PCR product detection & AGE</div>

<li><div class="text_font">making the agarose gel（see 2016.07.25 Morning)</div></li>

<li><div class="text_font">sample application：use micropipet to add 2μl DNA sample from experiment 1 to point template, and then add 1 μl buffer, mix ,then add it into the sample hole. Add 10 μl marker into one side the sample hole.</div></li>

<li><div class="text_font">electrophoresis:turn on the power,adjust the voltage. It can be observe after 30min.</div></li>

<li><div class="text_font">observation:put the gel on the ultraviolet detector, turn on the UV light, white nucleic acid strip can be seen. Based on the thickness and flosorescence intensity as well as the marker, we can estimate the concentration and molecular weight of DNA.</div></li>

<li><div class="text_font">result:the strip is very dull, and there are many spots around the picture</div></li>

<li><div class="text_font">analyze:</div>

<li><div class="text_font">We didn’t add add the reagent accurately, which led to not finishing the PCR that makes a low DNA concentration</div></li>

<li><div class="text_font">The dye wasn’t mixed with DNA completely, which makes the strip very dull</div></li>

<li><div class="text_font">The DNA extracted from the plants wasn’t pure, cause the appear of impurities</div></li>

<li><div class="text_font">For the gel was prepared a day ago, it may be polluted during the long time, which brought some impurities</div></li>

                                     <i class="fa fa-file-text"></i><div class="text_font"> 2016.7.27</div>

<small class="text-navy"><div class="text_font">Afternoon</div></small>

                                     <div class="m-b-xs text_font"><strong>the second time for PCR&AGE </strong></div>

<li><div class="text_font">experiment material and experiment apparatus is the as the first time(2016.07.26)</div></li>

<li><div class="text_font">experiment reagent :primers was decreased to two types(plf1 and hhl)</div></li>

<li><div class="text_font">experiment steps:</div>

<li><div class="text_font">PCR:the same as the first time (2016.07.26)</div></li>

<li><div class="text_font">sample application：use micropipet to add 2μl DNA sample from experiment 1 to point template, and then add 1 μl buffer, 0.5μl SYBY GREEN,mix ,wait for 10min,and then add it into the sample hole. Add 10 μl markertot o point template, 0.5μl SYBY GREEN,mix, wait for 5min, then add it into one side the sample hole.</div></li>

<li><div class="text_font"><font style="line-height:67%;font-size:83%">4. </font>is the same as the steps in the first time</div></li>

<li><div class="text_font">result:the brightness of strips are usual, but many strips’ length are less than 1000bp</div></li>

<li><div class="text_font">analyze:</div>

<li><div class="text_font">The primers may not be enough, they couldn’t combine with DNA perfectly, which led to the residue of primer,and didn’t get the target gene.</div></li>

<li><div class="text_font">When extracting DNA, some parts of the DNA may be enzymolysised by some DNA helicase, so the DNA can’t combine with the primers.</div></li>

                                     <i class="fa fa-file-text"></i><div class="text_font"> 2016.7.28</div>

<small class="text-navy"><div class="text_font">Afternoon</div></small>

                                     <div class="m-b-xs text_font"><strong>the third time for PCR&AGE </strong></div>

                                         <li><div class="text_font">experiment material and experiment apparatus is the as the first time(2016.07.26)</div></li>

<li><div class="text_font">experiment reagent :primers was decreased to two types(plf1 and hhl)</div></li>

<li><div class="text_font">experiment steps:</div>

<li><div class="text_font">PCR: the react sysytem is the same as the first time (2016.07.26), but the react settings was changed into:94℃×5min + （94℃×30s +60℃×30s + 72℃×2min）×30+ 72℃×10min + 4℃</div></li>

<li><div class="text_font">AGE: the same as the second time</div></li>

<li><div class="text_font">result: the brightness and length of strips are very usual,but there are some obvious entrainments</div></li>

<li><div class="text_font">Analyze:</div>

<li><div class="text_font">PCR might occur non-specificity amplification, which produced many small fragments</div></li>

<li><div class="text_font">The voltage of AGE might be too high, which damaged the aperture of agarose gel</div></li>

<li><div class="text_font">We might add too much primers</div></li>

<li><div class="text_font">We might didn’t add the dNTPs correctly, which led to the increasing of mispairing rate</div></li>