Team:ZJU-China/notebook.htm

From 2012.igem.org

(Difference between revisions)
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<p>2. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH5&alpha;), pCJDFB (DH5&alpha;) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.</p>
<p>2. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH5&alpha;), pCJDFB (DH5&alpha;) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.</p>
<p>3. Restriction enzyme digestion of D0-6-28-1, D0-6-28-2 and D0-6-28-3 with EcoR1 and Pst1.</p>
<p>3. Restriction enzyme digestion of D0-6-28-1, D0-6-28-2 and D0-6-28-3 with EcoR1 and Pst1.</p>
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<p><img src="http://igem.org/wiki/images/b/b4/Zju_notebook1.jpg"  width="80%"><p>
<p>It seems that we haven't achieved the correct plasmid but this may be caused by low concentration.</p>
<p>It seems that we haven't achieved the correct plasmid but this may be caused by low concentration.</p>
<h3>June 29</h3>
<h3>June 29</h3>
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<p>Name: Co-trans-7-07      Concentration: 9.7 ng/ul          A260/280: 1.79</p>
<p>Name: Co-trans-7-07      Concentration: 9.7 ng/ul          A260/280: 1.79</p>
<p>2. Make the first part K738000, RNA scaffold with promoter and terminator. Do PCR procedure.</p>
<p>2. Make the first part K738000, RNA scaffold with promoter and terminator. Do PCR procedure.</p>
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<p><img src="http://igem.org/wiki/images/b/b0/Zju_notebook2.jpg"  width="80%"><p>
<p>PCR of D0 seems to be correct, but there is nothing in pCJDFB and the plasmid of co-transformation bacteria is the same as that of pCJDD0.</p>
<p>PCR of D0 seems to be correct, but there is nothing in pCJDFB and the plasmid of co-transformation bacteria is the same as that of pCJDD0.</p>
<p>3. Re-transformation:</p>
<p>3. Re-transformation:</p>
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<h3>July 11</h3>
<h3>July 11</h3>
<p>1. Identify FA and FB with PCR.</p>
<p>1. Identify FA and FB with PCR.</p>
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<p><img src="http://igem.org/wiki/images/a/a2/Zju_notebook3.jpg"  width="80%"><p>
<p>2. Amplify FB-7-10-plate bacteria in liquid LB medium.</p>
<p>2. Amplify FB-7-10-plate bacteria in liquid LB medium.</p>
<h3>July 12</h3>
<h3>July 12</h3>
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<p>2. Amplify FB-7-10-plate1, FB-7-10-plate2, FA-6.29-liq (DH5&alpha;), FA-lix and FB-lix bacteria for minipreparing plasmids later.</p>
<p>2. Amplify FB-7-10-plate1, FB-7-10-plate2, FA-6.29-liq (DH5&alpha;), FA-lix and FB-lix bacteria for minipreparing plasmids later.</p>
<p>3. Identify FB with PCR again.We found there were problems with primers used in those reactions, thus leading to the failure.</p>
<p>3. Identify FB with PCR again.We found there were problems with primers used in those reactions, thus leading to the failure.</p>
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<p><img src="http://igem.org/wiki/images/9/9f/Zju_notebook4.jpg"  width="80%"><p>
<p>4. Ligation: D0 and pSB1C3, purified at July 08.</p>
<p>4. Ligation: D0 and pSB1C3, purified at July 08.</p>
<h3>July 13</h3>
<h3>July 13</h3>
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<p>Name: FA-7-13-lix      Concentration: 13.3 ng/ul        A260/280: 2.08</p>
<p>Name: FA-7-13-lix      Concentration: 13.3 ng/ul        A260/280: 2.08</p>
<p>Name: FB-7-13-yy      Concentration: 26.4 ng/ul        A260/280: 1.84</p>
<p>Name: FB-7-13-yy      Concentration: 26.4 ng/ul        A260/280: 1.84</p>
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<p><img src="http://igem.org/wiki/images/8/85/Zju_notebook5.jpg" width="80%"><p>
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<h3>July 14</h3>
<h3>July 14</h3>
<p>1. Co-transformation: </p>
<p>1. Co-transformation: </p>
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<p>Name: FB-7-14-5      Concentration: 33.1 ng/ul        A260/280: 0.68</p>
<p>Name: FB-7-14-5      Concentration: 33.1 ng/ul        A260/280: 0.68</p>
<p>Name: FB-7-14-6      Concentration: 26.1 ng/ul        A260/280: 1.64</p>
<p>Name: FB-7-14-6      Concentration: 26.1 ng/ul        A260/280: 1.64</p>
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<p><img src="http://igem.org/wiki/images/3/3e/Zju_notebook6.jpg"  width="80%"><p>
<p>3. Amplify glycerol stock bacteria co-transformation (BL21*(DE3)) on 3 resistance plates, glycerol stock bacteria D0 on Amp plate.</p>
<p>3. Amplify glycerol stock bacteria co-transformation (BL21*(DE3)) on 3 resistance plates, glycerol stock bacteria D0 on Amp plate.</p>
<p>4. Amplify FB-7-12-2, FB-7-12-4, FA-7-12-2-2 and FA-7-12-2-3 in LB liquid medium, named FB-7-14-2, FB-7-14-4, FA-7-14-2-2 and FA-7-14-2-3.</p>
<p>4. Amplify FB-7-12-2, FB-7-12-4, FA-7-12-2-2 and FA-7-12-2-3 in LB liquid medium, named FB-7-14-2, FB-7-14-4, FA-7-14-2-2 and FA-7-14-2-3.</p>
<p>5. Digest FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with Pst1.</p>
<p>5. Digest FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with Pst1.</p>
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<p><img src="http://igem.org/wiki/images/d/de/Zju_notebook7.jpg"  width="80%"><p>
<p>Maybe the concentration is too low that we can't recognize it.</p>
<p>Maybe the concentration is too low that we can't recognize it.</p>
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<p>2. Amplify glycerol stock D0 bacteria on two Amp plates, named D0-7-16-1 and D0-7-16-2.</p>
<p>2. Amplify glycerol stock D0 bacteria on two Amp plates, named D0-7-16-1 and D0-7-16-2.</p>
<p>3. Identify FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with PCR, new primers.</p>
<p>3. Identify FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with PCR, new primers.</p>
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<p><img src="http://igem.org/wiki/images/2/22/Zju_notebook8.jpg" width="80%"><p>
<p>The result of FA is not very ideal, while that of FB is not very exactly. The plasmid (all was Miniprep today) seems just stay where they were put in.</p>
<p>The result of FA is not very ideal, while that of FB is not very exactly. The plasmid (all was Miniprep today) seems just stay where they were put in.</p>
<h3>July 17</h3>
<h3>July 17</h3>
<p>1. Amplify FA-FB-7-15-90 and FA-FB-7-15-30 in LB liquid medium.</p>
<p>1. Amplify FA-FB-7-15-90 and FA-FB-7-15-30 in LB liquid medium.</p>
<p>2. Identify FA-7-13-2-2, FA-7-13-3-1 with PCR, increase Tm by 2Degrees Celsius.</p>
<p>2. Identify FA-7-13-2-2, FA-7-13-3-1 with PCR, increase Tm by 2Degrees Celsius.</p>
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<p><img src="http://igem.org/wiki/images/5/5f/Zju_notebook9.jpg"  width="80%"><p>
<p>3. Amplify glycerol stock co-transformation-2-1 bacteria on plate, named co3-7-17.</p>
<p>3. Amplify glycerol stock co-transformation-2-1 bacteria on plate, named co3-7-17.</p>
<h3>July 18</h3>
<h3>July 18</h3>
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<h3>July 21</h3>
<h3>July 21</h3>
<p>1. Gradient PCR to find an appropriate Tm value for FA.</p>
<p>1. Gradient PCR to find an appropriate Tm value for FA.</p>
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<p><img src="http://igem.org/wiki/images/2/2e/Zju_notebook10.jpg"  width="80%"><p>
<p>It seems that there is not a lot of differences.</p>
<p>It seems that there is not a lot of differences.</p>
<p>2. Amplify Co3-7-20 on plates, named Co3-7-21.</p>
<p>2. Amplify Co3-7-20 on plates, named Co3-7-21.</p>
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<p>1. Amplify Co3-7-21 in LB liquid medium, named Co3-7-22.</p>
<p>1. Amplify Co3-7-21 in LB liquid medium, named Co3-7-22.</p>
<p>2. Colony PCR to test Co2-7-21, Co3-7-22, and FB-7-19.</p>
<p>2. Colony PCR to test Co2-7-21, Co3-7-22, and FB-7-19.</p>
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<p><img src="http://igem.org/wiki/images/a/a8/Zju_notebook11.jpg"  width="80%"><p>
<p>We found that we may use a wrong protocol of colony PCR.</p>
<p>We found that we may use a wrong protocol of colony PCR.</p>
<p>3. Co-transform D0, FA and FB, named Co3-7-22. Co-transform FA and FB, named Co2-7-22.</p>
<p>3. Co-transform D0, FA and FB, named Co3-7-22. Co-transform FA and FB, named Co2-7-22.</p>
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<p>5. Amplify D0-7-23 plate in LB liquid medium.</p>
<p>5. Amplify D0-7-23 plate in LB liquid medium.</p>
<p>6. Colony PCR of FA, FB and Co3.</p>
<p>6. Colony PCR of FA, FB and Co3.</p>
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<p><img src="http://igem.org/wiki/images/1/1c/Zju_notebook12.jpg"  width="80%"><p>
<p>7. Miniprep plasmid of Co3-7-22-1 and Co3-7-22-2 for PCR procedure.</p>
<p>7. Miniprep plasmid of Co3-7-22-1 and Co3-7-22-2 for PCR procedure.</p>
<p>Name: Co3-7-23-1      Concentration: 29.9 ng/ul        A260/280: 1.91</p>
<p>Name: Co3-7-23-1      Concentration: 29.9 ng/ul        A260/280: 1.91</p>
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<p>3. Transform FA-7-23-2-3-1 and FB-7-24-2-4 into BL21*(DE3), named Co2-7-24.</p>
<p>3. Transform FA-7-23-2-3-1 and FB-7-24-2-4 into BL21*(DE3), named Co2-7-24.</p>
<p>4. PCR to test (1) if pCJDD0 reacts with primer FAF and FAR; (2) further confirm the success of co-transformation.</p>
<p>4. PCR to test (1) if pCJDD0 reacts with primer FAF and FAR; (2) further confirm the success of co-transformation.</p>
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<p><img src="http://igem.org/wiki/images/c/c2/Zju_notebook13.jpg"  width="80%"><p>
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<p><img src="http://igem.org/wiki/images/d/df/Zju_notebook14.jpg"  width="80%"><p>
<p>Unfortunately, we found we can achieve the same result of PCR whatever the template is if we use the same pare of primers. In contrast, we can't find there is any similarity between template and primers. We are really confused about that. It means that we can't test the success of co-transformation by PCR.</p>
<p>Unfortunately, we found we can achieve the same result of PCR whatever the template is if we use the same pare of primers. In contrast, we can't find there is any similarity between template and primers. We are really confused about that. It means that we can't test the success of co-transformation by PCR.</p>
<p>5. Amplify FB-7-23 bacteria in LB liquid medium.</p>
<p>5. Amplify FB-7-23 bacteria in LB liquid medium.</p>
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<p>Name: K738000-7-30-10      Concentration: 43.6 ng/ul        A260/280: 1.98</p>
<p>Name: K738000-7-30-10      Concentration: 43.6 ng/ul        A260/280: 1.98</p>
<p>PCR of K738000-7-30</p>
<p>PCR of K738000-7-30</p>
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<p><img src="http://igem.org/wiki/images/d/df/Zju_notebook15.jpg"  width="80%"><p>
<p>Unfortunately, there was a slight electrophoretic band in control. But the sequencing result came later verified that we've got the correct ligation product.</p>
<p>Unfortunately, there was a slight electrophoretic band in control. But the sequencing result came later verified that we've got the correct ligation product.</p>
<p>2. Pick up single colonies from plate of FB-7-26-2 and amplify it in LB liquid medium.</p>
<p>2. Pick up single colonies from plate of FB-7-26-2 and amplify it in LB liquid medium.</p>
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<p>Name: K738000-7-30-11      Concentration: 46.0 ng/ul        A260/280: 1.97</p>
<p>Name: K738000-7-30-11      Concentration: 46.0 ng/ul        A260/280: 1.97</p>
<p>Name: FA-7-28-1            Concentration: 33.0 ng/ul        A260/280: 2.04</p>
<p>Name: FA-7-28-1            Concentration: 33.0 ng/ul        A260/280: 2.04</p>
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<p><img src="http://igem.org/wiki/images/e/e6/Zju_notebook16.jpg"  width="80%"><p>
<h3>August 01</h3>
<h3>August 01</h3>
<p>Double digest K738000-7-23-2 and K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.</p>
<p>Double digest K738000-7-23-2 and K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.</p>
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<p>Name: FA-8-4        Concentration: 19.6 ng/ul        A260/280: 1.97</p>
<p>Name: FA-8-4        Concentration: 19.6 ng/ul        A260/280: 1.97</p>
<p>Name: FB-8-4        Concentration: 35.9 ng/ul        A260/280: 1.98</p>
<p>Name: FB-8-4        Concentration: 35.9 ng/ul        A260/280: 1.98</p>
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<p><img src="http://igem.org/wiki/images/c/cc/Zju_notebook17.jpg"  width="80%"><p>
<p>Unfortunately, the PCR results were so different from that of original plasmids. We couldn't figure out the reason.</p>
<p>Unfortunately, the PCR results were so different from that of original plasmids. We couldn't figure out the reason.</p>
<p>2. Amplify Venus-8-2 in LB liquid medium, named Venus-8-4.</p>
<p>2. Amplify Venus-8-2 in LB liquid medium, named Venus-8-4.</p>
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<p>1. Pick up single colonies from plate of FAM-8-4 and FBP-8-4 and amplify it in LB liquid medium and LB plates.</p>
<p>1. Pick up single colonies from plate of FAM-8-4 and FBP-8-4 and amplify it in LB liquid medium and LB plates.</p>
<p>2. Colony PCR of FAM-8-4-2 and FBP-8-4-2</p>
<p>2. Colony PCR of FAM-8-4-2 and FBP-8-4-2</p>
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<p><img src="http://igem.org/wiki/images/c/c8/Zju_notebook18.jpg"  width="80%"><p>
<p>3. Double digest BB-8-4-1 with (1) EcoR1 and Pst1, (2) Xba1 and Spe1, get linear backbone through PCR cleanup kit.</p>
<p>3. Double digest BB-8-4-1 with (1) EcoR1 and Pst1, (2) Xba1 and Spe1, get linear backbone through PCR cleanup kit.</p>
<p>4. Miniprep plasmid of Venus-8-4.</p>
<p>4. Miniprep plasmid of Venus-8-4.</p>
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<p>1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.</p>
<p>1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.</p>
<p>2. PCR of part K738001:</p>
<p>2. PCR of part K738001:</p>
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<p><img src="http://igem.org/wiki/images/0/0a/Zju_notebook19.jpg"  width="80%"><p>
<p>3. PCR of original pCJDFA and pCJDFB.</p>
<p>3. PCR of original pCJDFA and pCJDFB.</p>
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<p><img src="http://igem.org/wiki/images/f/f3/Zju_notebook20.jpg"  width="80%"><p>
<p>4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.</p>
<p>4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.</p>
<p>Name: FAM-8-6-1        Concentration: 56.7 ng/ul          A260/280: 1.95</p>
<p>Name: FAM-8-6-1        Concentration: 56.7 ng/ul          A260/280: 1.95</p>
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<p>PCR1: delete 3bp after MS2 in D0.</p>
<p>PCR1: delete 3bp after MS2 in D0.</p>
<p>PCR2: delete 3bp before MS2 in D0.</p>
<p>PCR2: delete 3bp before MS2 in D0.</p>
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<p><img src="http://igem.org/wiki/images/9/94/Zju_notebook21.jpg" width="80%"><p>
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<p><img src="http://igem.org/wiki/images/b/bb/Zju_notebook22.jpg"  width="80%"><p>
<h3>August 10</h3>
<h3>August 10</h3>
<p>1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.</p>
<p>1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.</p>
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<p>3. Mutation of D0: </p>
<p>3. Mutation of D0: </p>
<p>PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.</p>
<p>PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.</p>
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<p><img src="http://igem.org/wiki/images/7/7c/Zju_notebook23.jpg"  width="80%"><p>
<p>PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.</p>  
<p>PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.</p>  
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<p><img src="http://igem.org/wiki/images/6/63/Zju_notebook24.jpg"  width="80%"><p>
<p>PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.</p>
<p>PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.</p>
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<p><img src="http://igem.org/wiki/images/7/7d/Zju_notebook25.jpg"  width="80%"><p>
<p>4. Amplify FB-8-7 in LB liquid medium.</p>
<p>4. Amplify FB-8-7 in LB liquid medium.</p>
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<p>LIU Xiao comes to carry out point mutation experiment to modify origin D0 in PCR method. He intended to build a library. However, we have only one month left, too short to finish the library.</p>
<p>LIU Xiao comes to carry out point mutation experiment to modify origin D0 in PCR method. He intended to build a library. However, we have only one month left, too short to finish the library.</p>
<p>Yan's fluorescent test with part K537009 is modestly successful. Most excitingly, Yan and Chen capture a love heart (or you can refer to it as a smiling smile) imaging by E.coli.</p>
<p>Yan's fluorescent test with part K537009 is modestly successful. Most excitingly, Yan and Chen capture a love heart (or you can refer to it as a smiling smile) imaging by E.coli.</p>
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<p><img src="http://igem.org/wiki/images/d/d2/Zju_notebook26.jpg"  width="80%"><p>
<h3>2012/8/14, Tuesday 14, 2012</h3>
<h3>2012/8/14, Tuesday 14, 2012</h3>
<p>Chen makes competent cell with BL21 star DE3 borrow from Zhejiang Sci-Tech University.</p>
<p>Chen makes competent cell with BL21 star DE3 borrow from Zhejiang Sci-Tech University.</p>
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<p>Yan tried the parts of Theophylline riboswitch with YFP Venus reporter and it worked.  
<p>Yan tried the parts of Theophylline riboswitch with YFP Venus reporter and it worked.  
(Though excite 515nm emit 528 mn is too close for Synergy hybrid reader to detect and we change it into 505nm and 535nm.)</p>
(Though excite 515nm emit 528 mn is too close for Synergy hybrid reader to detect and we change it into 505nm and 535nm.)</p>
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<p><img src="http://igem.org/wiki/images/b/b4/Zju_notebook27.jpg"  width="80%"><p>
<p>You can see the fluorescence intensity increasing with Theophylline concentration.</p>
<p>You can see the fluorescence intensity increasing with Theophylline concentration.</p>
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<p>He makes a new RNA scaffold, Nucleic Acid Electrophoresis as follows. </p>
<p>He makes a new RNA scaffold, Nucleic Acid Electrophoresis as follows. </p>
<p>First step:</p>
<p>First step:</p>
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<p><img src="http://igem.org/wiki/images/2/29/Zju_notebook28.jpg" width="80%"><p>
<p>Second step:</p>
<p>Second step:</p>
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<p><img src="http://igem.org/wiki/images/7/7d/Zju_notebook29.jpg" width="80%"><p>
<p>First step product is due in 50bp and second is due in 200bp.
<p>First step product is due in 50bp and second is due in 200bp.
But the Electrophoresis effect is not good.</p>
But the Electrophoresis effect is not good.</p>
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<p>1. K411003 (Taipei 2010) were adding a series of Theophylline. This experiment is used both for testing parts and fishing for the condition of our synthetic D0 from Genescript's future GFP experiment. </p>
<p>1. K411003 (Taipei 2010) were adding a series of Theophylline. This experiment is used both for testing parts and fishing for the condition of our synthetic D0 from Genescript's future GFP experiment. </p>
<p>2. The Synergy hybrid reader data is good. You can see the fluorescence intensity increasing with Theophylline concentration.</p>
<p>2. The Synergy hybrid reader data is good. You can see the fluorescence intensity increasing with Theophylline concentration.</p>
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<p><img src="http://igem.org/wiki/images/b/bb/Zju_notebook30.jpg" width="80%"><p>
<h3>2012/8/22 Wednesday, August 22, 2012</h3>
<h3>2012/8/22 Wednesday, August 22, 2012</h3>
<p>1. 00:39 in the morning, YU Jianing sends an e-mail to Yan from her hometown, attaching version 1 ODE modeling and some questions about biology meanings.</p>
<p>1. 00:39 in the morning, YU Jianing sends an e-mail to Yan from her hometown, attaching version 1 ODE modeling and some questions about biology meanings.</p>
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<h3>2012/8/27 Monday, August 27, 2012</h3>
<h3>2012/8/27 Monday, August 27, 2012</h3>
<p>1.  Chen did the overlap PCR and construct FB-2X-PP7 (approximately 630 bp). It seems correct in the agarose gel electrophoresis. (1000 marker on the left and 5000 marker on the right)</p>
<p>1.  Chen did the overlap PCR and construct FB-2X-PP7 (approximately 630 bp). It seems correct in the agarose gel electrophoresis. (1000 marker on the left and 5000 marker on the right)</p>
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<p><img src="http://igem.org/wiki/images/2/24/Zju_notebook31.jpg"  width="80%"><p>
<p>2.  Yan and Chen PCR MS2 from plasmids Dr. Delebecque sent us respectively.
<p>2.  Yan and Chen PCR MS2 from plasmids Dr. Delebecque sent us respectively.
Yan uses 57.5 Degrees Celsius as melting temperature and Chen uses 58.5 and 63.5.
Yan uses 57.5 Degrees Celsius as melting temperature and Chen uses 58.5 and 63.5.
But the electrophoretic bands seem incorrect.
But the electrophoretic bands seem incorrect.
(Next day we find angrily that the primer the company synthesized is wrong!)</p>
(Next day we find angrily that the primer the company synthesized is wrong!)</p>
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<p><img src="http://igem.org/wiki/images/a/a1/Zju_notebook32.jpg"  width="80%"><p>
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<p><img src="http://igem.org/wiki/images/9/95/Zju_notebook33.jpg"  width="80%"><p>
<h3>2012/8/28 Tuesday, August 28, 2012</h3>
<h3>2012/8/28 Tuesday, August 28, 2012</h3>
<p>1.  LIU uses gel extraction to get last day Chen's MS2 and carry on overlap PCR to make FA-2X-MS2.</p>
<p>1.  LIU uses gel extraction to get last day Chen's MS2 and carry on overlap PCR to make FA-2X-MS2.</p>

Revision as of 03:47, 19 September 2012

NOTEBOOK

01 BRAINSTORM

02 PROTOCAL

03 LAB NOTE