Team:SDU-Denmark/labnotes7

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== Photosensor ==
== Photosensor ==
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=== Insertion of B0015 in pSB3C5 and pSB3T5 ===
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'''Date:''' 8/23 - 8/29 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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==== Pfu PCR amplification and purification of B0015 ====
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'''Date:''' 8/23 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]<br>
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'''Notes:'''<br>
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4 PCR reactions are prepared. 2uL Miniprep of pSB1AK3 (white 43) are distrubuted in each tube. PCR tubes are marked B00153.A-D.<br>
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Premix x5:<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>pfu buffer + MgSO<small>4</small></td>
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      <td>25uL</td>
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    </tr>
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    <tr>
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      <td>dNTP mix</td>
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      <td>7.5uL</td>
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    </tr>
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    <tr>
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      <td>VF2 primer</td>
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      <td>7.5uL</td>
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    </tr>
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    <tr>
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      <td>VR primer</td>
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      <td>7.5uL</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>190uL</td>
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    </tr>
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    <tr>
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      <td>pfu polymerase&nbsp;</td>
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      <td>2uL</td>
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    </tr>
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</table><br><br>
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PCR program:<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>start</td>
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      <td>94C</td>
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      <td>3min</td>
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    </tr>
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    <tr>
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      <td>denaturating</td>
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      <td>94C</td>
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      <td>2min</td>
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    </tr>
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    <tr>
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      <td>annealing</td>
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      <td>55C</td>
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      <td>30s</td>
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    </tr>
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    <tr>
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      <td>elongation</td>
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      <td>72C</td>
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      <td>30s</td>
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    </tr>
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    <tr>
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      <td>go to</td>
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      <td>2</td>
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      <td>rep.29x</td>
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    </tr>
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    <tr>
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      <td>end</td>
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      <td>72C</td>
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      <td>5min</td>
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    </tr>
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    <tr>
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      <td>hold</td>
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      <td>4C</td>
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      <td></td>
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    </tr>
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</table><br>
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5uL of the PCR product was loaded onto a 2% agarose gel. Gene ruler 100bp DNA ladder was used ad marker.<br><br>
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'''Results:'''<br>
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'''Analysis:'''<br>
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A strong band was observed at app. 450bp, and the rest of the PCR product was purified according to protocol using the GFX purification kit.<br>
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End conc.: 48ng/uL <br><br>
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==== Restriction digest of B0015 ====
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'''Date:''' 8/24 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 B0015:<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>38uL</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>8uL</td>
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    </tr>
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    <tr>
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      <td>EcoRI</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>B0015</td>
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      <td>30uL</td>
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    </tr>
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</table><br><br>
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The digested sample was loaded onto a 2% agarose extraction gel. Uncut B0015 was used as controle. Gene ruler 100bp DNA ladder was used as marker.<br>
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DNA was extracted from gel according to protocol.<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>B0015</td>
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      <td>6.5</td>
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    </tr>
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    <tr>
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      <td>pSB3C5</td>
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      <td>50.8</td>
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    </tr>
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    <tr>
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      <td>pSB3T5</td>
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      <td>9.9</td>
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    </tr>
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</table><br><br>
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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 and pSB1C3 and pCB1AK3 ====
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'''Date:''' 8/24 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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For each of the ligations three ligation mixtures were prepared. vector concentrations of 10n0g/uL (pSB3C5) and 50ng/uL (pSB3T5) respectively was used for each mixture. Appropiate amount of insert was added to reach vector:insert ratios of 1:1, 1:3 and 1:6 respectively. <br>
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Ligation mixtures (B0015 in pSB3C5):<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>pSB3C5</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>B0015 </td>
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      <td>0.7uL</td>
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      <td>2uL</td>
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      <td>4.5uL</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>14.3uL</td>
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      <td>13uL</td>
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      <td>10.5uL</td>
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    </tr>
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</table><br>
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Ligation mixtures (B0015 in pSB3T5):<br>
 +
<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
 +
    <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>5uL</td>
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      <td>5uL</td>
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      <td>5uL</td>
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    </tr>
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    <tr>
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      <td>B0015 </td>
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      <td>0.5uL</td>
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      <td>1uL</td>
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      <td>2uL</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>11.5uL</td>
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      <td>11uL</td>
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      <td>10uL</td>
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    </tr>
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</table><br><br>
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The samples was 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:''' 8/25 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 there were many colonies on plates with cells transformed with either of the ligation mixtures..<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]] 13:41, 26 September 2010 (UTC)<br><br>
=== Colony PCR on B0017 ===
=== Colony PCR on B0017 ===
Date: 27/8<br>
Date: 27/8<br>

Revision as of 15:37, 26 September 2010