Team:OUC-China/Result/week11

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<div id="side">
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<ul class="sidebar">
<ul class="sidebar">
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<li><a href="/Team:OUC-China/Result/summary">Notebook - Summary</a></li>
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<li><a href="/Team:OUC-China/Result/Notebook">Notebook - Summary</a></li>
                 <ul>
                 <ul>
                   <li><a href="/Team:OUC-China/Result/week1">Week 1</a></li>
                   <li><a href="/Team:OUC-China/Result/week1">Week 1</a></li>
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                 </ul>
                 </ul>
<li><a href="/Team:OUC-China/Result/date">Data</a></li>
<li><a href="/Team:OUC-China/Result/date">Data</a></li>
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              <li><a href="/Team:OUC-China/Result/Puf2011d">Previous Biobricks</a></li>
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<h2>9-12</h2>
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<h2>9-19</h2>
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<p>Since there was only one of the three tubes of 6&leuB turned turbid, we took some bacteria from it for reservation and examination. The rate of A260/A280 was not normal and the result of gel electrophoresis was not satisfactory, either.</p>
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<p>Enlargement culture the 6&leu ligated in 3A method at 11 a.m. and coat them on the square plates then cultured in incubator.</p>
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<p>There were still no colonies on plat solid medium so we repeated the ligation today.</p>
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<p>After electrophoresis of the no.6 and the carrier C and cutting the gels, we recover them in gel extraction mini kit. at 12 a.m.  The carrier C is uesd for standardization.</p>
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<p>We conducted enzyme digestion of No.21 and No.22 for future ligation with the digested product of No.3.</p>
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  <p>Put E0020,C0076 and praiI in LB again. Disinfect 7 test tubes.</p>
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<p>In the afternoon we tested loop ligation by signal molecular. For loop II, we used lux&las and lux(control group) as inducer. For loop III, we used lux&las&cin and lux&las(control group). Tomorrow we will get the result by observing under fluorescent microscope.</p>
+
  <p>We side the concentration of recovered 6 and leu. But they are too slow to be concentrated. After being concentrated, no.6’s concentration has been to 30, but leu is the same. So we prepare to cut Leu again.</p>
-
<h2>9-13</h2>
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  <p>In the evening, we side the concentration of 13 parts which have been standardized yesterday.</p>
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<p>Today we repeated PCR process using yesterday’s templates (20 samples, 10 pairs of part). But the gel electrophoresis all showed smears.</p>
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<h2>9-20</h2>
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<p>We selected 21&3, 22&3, 8&6 to conduct the transformation, coat-cultured the product, continued the ligation with PSB1C3 vector and large scale of gel recollection.</p>
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  <p>Ligating 6&leu in 3A method failed. Leu are double-enzyme cutd to be ligated with no.6 in traditional method.</p>
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<p>The result of screening of the signal-molecular-induced product showed that the loop of lux&las in loop II appeared weak fluorescence. The lux-reduced loop II did not show fluorescence (control group). We supposed that there was something wrong with No.16 fluorescent protein and decided to re-build loop II.</p>
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  <p>C0076,E0020 and praiI are preservated and isolated at 1 p.m.</p>
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<p>Since the bacilli did not turn turbid, we re-inoculated the single colony from dish-cultured loop III bacteria to LB medium.</p>
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<h2>9-21</h2>
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<h2>9-14</h2>
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  <p>Make 6&leu which were ligated yesterday heated to be inactivited and transformed and then coat them on square plates.</p>
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<p>After 20 hours, the parts—21&3,22&3,8&6 haven’t grown out, but test tube with circuit-III’s have been turbid.</p>
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  <p>Put raiR1、raiR2、raiR3、raiI1、raiI2、sinR1、sinR2、sinR3 in EP tubes to shake culturing to be used in sequencing by PCR.</p>
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<p>Ligate the parts—sinR,sinI and pro(sin) with the carrier C to be standardized.</p>
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  <p>Asparaguspea are cultured in shaking bed at 30℃ waiting for PCR.</p>
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<p>By detecting, the part pro-RBS-sinR’s electrophoretic band is too shallow, and the part pro-RBS-raiI, pro-RBS-raiR and pro-RBS-sinI’s bands are all blended, and the sinR has less blended bands. We retrieve the sinR,sinRpro,raiI-pro,sinI-pro and lay down the others.</p>
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  <p>Put the primer ordered in EP tubes.</p>
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<p>The part NO.16 in Circuit-II is proved that its length doesn’t accord with the standerd.We suspect it’s the part’s questions. Then we decided to ligate the NO.16 with small parts composed the NO.16. Today C0076 and E0020 have been transformed.</p>
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<h2>9-22</h2>
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<h2>9-15</h2>
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  <p>6&leu’s plates have come out bacteria colonies. Pick out some in LB.</p>
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<p>The standardized five parts are transformed at 10 a.m. and coated on square plate at 2 p.m.</p>
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  <p>Enzyme cut E0020,C0076 and raiI1,2,3. but we find we use the wrong enzyme. We do it again in the afternoon.</p>
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<p> 6&leu’s first test tube is proved to be ligated correctly.</p>
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  <p>Amplifications by PCR: rhiI、rhiR、rhiABCpro、rhiIpro、pro-RBS-rhiI.</p>
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<p> 21&3 and 22&3’s bacteria colony haven’t appeared on the plates.So we have to isolate the plasmids again, and put them in LB culture medium.</p>
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  <p>Verify by PCR: raiR1、raiR2、raiR3、raiI1、raiI2、sinR1、sinR2、sinR3. Then verify them in electrophoresis.</p>
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<p> 8&6 are enlargement cultured again. 6&len are double-enzyme cutd in the evening to be ligated in the traditional method and 3A method.</p>
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<h2>9-23</h2>
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<p> We pick ou the bacteria colony of E0020 and C0076, and put them in LB culture medium with shake cultivation at 37℃.</p>
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  <p>Extract raiI,raiR,raiABCpro. The parts—raiIpro,pro-RBS-raiI which were fail to be synthetized keep on PCR. So does the parts which were verified in PCR.</p>
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<h2>9-17</h2>
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  <p>Extracte the plasmids of 6&leu, and they’re correct verified by enzyme cut.</p>
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<p>After detecting Leu,we find the plasmid in no.2 and no.3 tude still can be used. Take some bacteria in LB culture medium. </p>
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  <p>LB square plates with C+ have lost efficacy. So we make up medium with antibiotics.</p>
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<h2>9-18</h2>
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  <p>Double-enzyme cut with EcoRI and PstI: raiI、raiR、raiABCpro、18-3-11、14-19、circuit-I、5&4、14-19-13 and carrier C.</p>
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<p>Detect leu and 6 which have been cut and then ligate them in 3A method.</p>
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  <p>Throw away some square plates with blended bacteria after being desinfected.</p>
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<p>  Cut 6 for the second time at 5:40 p.m. to ligate with leu in 3A method.</p>
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<h2>9-24</h2>
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<p>  Keep on standardize parts including raiR1,raiR2,raiR3,raiI,raiI2,sinR1,sinR2,sinR3,sinI1,sinI2,sinI3,psinI1,psinI2.</p>
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  <p>6&leu have been ligated successfully. From today, we begin to finish the circuit-I,II,III to put the Leu in the circuits.</p>
 +
  <p>Enzyme cut 5-4,6&leu,18-3-11,13,carrier AC, carrier AK, C0076 and 6. extracte 6 and carrier AK. Ligate 18-3-11&eu-6；13&leu-6；C0076&6.</p>
 +
  <p>Activate the strain of 5-4,3,E0020 from preservation tubes.</p>
 +
  <p>Clear up preservation tubes. Throw away raiR2、raiI1、sinR3。SinI1、2、3；psinI1、2. Put raiIpro1,2,3 in EP tubes to be cultured and then be sequenced with sinR1、2；raiI2、raiR1.</p>
 +
  <p>PCR rhiI、rhiR、rhiABCpro, rhiIpro、pro-RBS-rhiI.</p>
 +
  <p>Standardizations: ligate carrier C separately with circuit-I,18-3-11,14-19,5-4,14-19-13.</p>
 +
<h2>9-25</h2>
 +
<p>The plates which are used for standardizing 14-19,circuit-I and 18-3-11 aren’t all successful. Only the 14-19 plates have bacteria colony. Pick out the bacteria colony of14-19 and RFP into LB.  Circuit-I ,18-3-11 and 14-19 are transformed again. In the afternoon,we also transform some parts-- rhiR、rhiABCpro、5&4、18-3-11&leu-6、13&leu-6、C0076&6, and coat them on the square plates.</p>
 +
<p>  Recover the product—rhiI by PCR and ligate it and rhiR, LeuB with carrier C to be standardized. </p>
 +
<p>  In the evening, we standardized the parts—leuB,14-19-13,rhiR and RFP.</p>
 +
<p>  Extract the plasmids of 5-4, 3 and E0020 activated yesterday.</p>
 +
<p>  Ligate E0020 with 6.</p>
 +
<p>  We transforme the part-J13002 and coat them on square plates.</p>
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Latest revision as of 02:38, 29 October 2011

9-19

Enlargement culture the 6&leu ligated in 3A method at 11 a.m. and coat them on the square plates then cultured in incubator.

After electrophoresis of the no.6 and the carrier C and cutting the gels, we recover them in gel extraction mini kit. at 12 a.m. The carrier C is uesd for standardization.

Put E0020,C0076 and praiI in LB again. Disinfect 7 test tubes.

We side the concentration of recovered 6 and leu. But they are too slow to be concentrated. After being concentrated, no.6’s concentration has been to 30, but leu is the same. So we prepare to cut Leu again.

In the evening, we side the concentration of 13 parts which have been standardized yesterday.

9-20

Ligating 6&leu in 3A method failed. Leu are double-enzyme cutd to be ligated with no.6 in traditional method.

C0076,E0020 and praiI are preservated and isolated at 1 p.m.

9-21

Make 6&leu which were ligated yesterday heated to be inactivited and transformed and then coat them on square plates.

Put raiR1、raiR2、raiR3、raiI1、raiI2、sinR1、sinR2、sinR3 in EP tubes to shake culturing to be used in sequencing by PCR.

Asparaguspea are cultured in shaking bed at 30℃ waiting for PCR.

Put the primer ordered in EP tubes.

9-22

6&leu’s plates have come out bacteria colonies. Pick out some in LB.

Enzyme cut E0020,C0076 and raiI1,2,3. but we find we use the wrong enzyme. We do it again in the afternoon.

Amplifications by PCR: rhiI、rhiR、rhiABCpro、rhiIpro、pro-RBS-rhiI.

Verify by PCR: raiR1、raiR2、raiR3、raiI1、raiI2、sinR1、sinR2、sinR3. Then verify them in electrophoresis.

9-23

Extract raiI,raiR,raiABCpro. The parts—raiIpro,pro-RBS-raiI which were fail to be synthetized keep on PCR. So does the parts which were verified in PCR.

Extracte the plasmids of 6&leu, and they’re correct verified by enzyme cut.

LB square plates with C+ have lost efficacy. So we make up medium with antibiotics.

Double-enzyme cut with EcoRI and PstI: raiI、raiR、raiABCpro、18-3-11、14-19、circuit-I、5&4、14-19-13 and carrier C.

Throw away some square plates with blended bacteria after being desinfected.

9-24

6&leu have been ligated successfully. From today, we begin to finish the circuit-I,II,III to put the Leu in the circuits.

Enzyme cut 5-4,6&leu,18-3-11,13,carrier AC, carrier AK, C0076 and 6. extracte 6 and carrier AK. Ligate 18-3-11&eu-6；13&leu-6；C0076&6.

Activate the strain of 5-4,3,E0020 from preservation tubes.

Clear up preservation tubes. Throw away raiR2、raiI1、sinR3。SinI1、2、3；psinI1、2. Put raiIpro1,2,3 in EP tubes to be cultured and then be sequenced with sinR1、2；raiI2、raiR1.

PCR rhiI、rhiR、rhiABCpro, rhiIpro、pro-RBS-rhiI.

Standardizations: ligate carrier C separately with circuit-I,18-3-11,14-19,5-4,14-19-13.

9-25

The plates which are used for standardizing 14-19,circuit-I and 18-3-11 aren’t all successful. Only the 14-19 plates have bacteria colony. Pick out the bacteria colony of14-19 and RFP into LB. Circuit-I ,18-3-11 and 14-19 are transformed again. In the afternoon,we also transform some parts-- rhiR、rhiABCpro、5&4、18-3-11&leu-6、13&leu-6、C0076&6, and coat them on the square plates.

Recover the product—rhiI by PCR and ligate it and rhiR, LeuB with carrier C to be standardized.

In the evening, we standardized the parts—leuB,14-19-13,rhiR and RFP.

Extract the plasmids of 5-4, 3 and E0020 activated yesterday.

Ligate E0020 with 6.

We transforme the part-J13002 and coat them on square plates.

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