Team:SDU-Denmark/labnotes10

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(Lab notes (9/13 - 9/19))
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In the lanes containing PCR product of colonies transformed with pKJ606 bands at app. 2000bp are observed, indicating that the colonies contains the right plasmids. No bands was observed in lanes containing PCR product of colonies transformed with pSB3T5, and this experiment needs to be redone.<br>
In the lanes containing PCR product of colonies transformed with pKJ606 bands at app. 2000bp are observed, indicating that the colonies contains the right plasmids. No bands was observed in lanes containing PCR product of colonies transformed with pSB3T5, and this experiment needs to be redone.<br>
--[[User:Tipi|Tipi]] 06:25, 28 September 2010 (UTC)<br><br>
--[[User:Tipi|Tipi]] 06:25, 28 September 2010 (UTC)<br><br>
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=== Insertion of PS + double terminator in pSB3T5 with J13002 (no.2) ===
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'''Date:''' 9/16 - 9/19 2010<Br>
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'''Done By:''' Maria and Lc<Br>
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'''Protocol:''' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1][https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.3 DE1.3][https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1][https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.2 LG1.2][https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1 CC1.1][https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1][https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3 CP1.3]<Br>
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==== Restriction digest of PS + double terminator and PSB3T5 ====
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'''Date:''' 9/17 2010<Br>
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'''Done By:''' Maria and Lc<Br>
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'''Protocol:'''[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1][https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.3 DE1.3]<br>
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'''Notes:'''<br>
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Restriction mixture pSB3T5:<br>
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<table style="text-align: left;" border="1"
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  cellpadding="2" cellspacing="2">
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    <tr>
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      <td>H<small>2</small>O</td>
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      <td>14uL</td>
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    </tr>
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    <tr>
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      <td>FD green buffer</td>
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      <td>4uL</td>
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    </tr>
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    <tr>
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      <td>SpeI</td>
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      <td>2uL</td>
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    </tr>
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    <tr>
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      <td>PstI</td>
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      <td>2uL</td>
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    </tr>
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    <tr>
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      <td>pSB3T5</td>
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      <td>20uL</td>
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    </tr>
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</table><br><br>
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Restriction mixture PS + double terminator:<br>
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>H<small>2</small>O</td>
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      <td>38uL</td>
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    </tr>
 +
    <tr>
 +
      <td>FD green buffer</td>
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      <td>8uL</td>
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    </tr>
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    <tr>
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      <td>XbaI</td>
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      <td>4uL</td>
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    </tr>
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    <tr>
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      <td>PstI</td>
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      <td>4uL</td>
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    </tr>
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    <tr>
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      <td>PS + double terminator</td>
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      <td>30uL</td>
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    </tr>
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</table><br><br>
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Samples were digested for 10min. at 37C.<br>
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Digested samples were loaded onto a 1.5% agarose extraction gel. Uncut PS + double terminator and pSB3T5 were used as controles. Gene ruler DNA ladder mix was used as marker.<br>
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Loading scheme:<br>
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td>Lane</td>
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      <td>sample</td>
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    </tr>
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    <tr>
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      <td>1</td>
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      <td>Marker</td>
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    </tr>
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    <tr>
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      <td>2</td>
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      <td>pSB3T5</td>
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    </tr>
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    <tr>
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      <td>3</td>
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      <td>uncut pSB3T5</td>
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    </tr>
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    <tr>
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      <td>4</td>
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      <td>PS + double terminator</td>
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    </tr>
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<tr>
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      <td>5</td>
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      <td>uncut PS + double terminator</td>
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    </tr>
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</table><br>
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DNA was extracted from gel, according to protocol. Samples were diluted in 20uL H2O.<br><br>
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'''Results:'''<br>
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DNA conc:
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td>sample</td>
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      <td>conc. (ng/uL)</td>
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    </tr>
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    <tr>
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      <td>PS + double terminator</td>
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      <td>26.3</td>
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    </tr>
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    <tr>
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      <td>pSB3T5</td>
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      <td>25.8</td>
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    </tr>
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</table>
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'''Analysis:'''
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the purified DNA was used for ligation.<br><br>
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==== Ligation of PS + double terminator and pSB3T5 ====
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'''Date:''' 9/17 2010<Br>
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'''Done By:''' Maria and Lc<Br>
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'''Protocol:'''[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.2 LG1.2]<br>
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'''Notes:'''<br>
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Three ligation mixtures was prepared. 50ng of vector was used for each mixture. Appropiate amount of insert was added to reac vector:insert ratios of 1:1, 1:2 and 1:4 respectively. <br>
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Ligation mixtures (PS + double terminator in pSB3T5):<br>
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td></td>
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      <td>L1</td>
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      <td>L2</td>
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      <td>L3</td>
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    </tr>
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    <tr>
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      <td>T4 ligase buffer</td>
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      <td>2uL</td>
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      <td>2uL</td>
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      <td>2uL</td>
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    </tr>
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    <tr>
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      <td>T4 ligase</td>
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      <td>1uL</td>
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      <td>1uL</td>
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      <td>1uL</td>
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    </tr>
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    <tr>
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      <td>pSB3T5</td>
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      <td>2uL</td>
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      <td>2uL</td>
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      <td>2uL</td>
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    </tr>
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    <tr>
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      <td>PS + double terminator </td>
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      <td>1uL (2. dilution)</td>
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      <td>2.5uL</td>
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      <td>5uL</td>
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    </tr>
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    <tr>
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      <td>H<small>2</small>0</td>
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      <td>14uL</td>
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      <td>12.5uL</td>
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      <td>10uL</td>
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    </tr>
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</table><br>
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The samples were incubated at 17C ON at used for transformation<br><br>
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==== Transfomation of ligated plasmid in Top 10 E.coli ====
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'''Date:''' 9/15 2010<Br>
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'''Done By:''' Maria and Lc<Br>
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'''Protocol:'''[https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1 CC1.1][https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1]<br>
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'''Notes:'''<br>
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The compotent cells and transformation was carried out according to protocol.<br><br>
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'''Results:'''<br>
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the controle plates were okay, and colonies of different sizes was observed on plates of cells transformed with ligations.<br><br>
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'''Analysis:'''<br>
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The transformation was successfull and colonies was selected and used in coloni PCR.<br>
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--[[User:Tipi|Tipi]] 11:21, 28 September 2010 (UTC)<br><br>

Revision as of 11:21, 28 September 2010