Team:SDU-Denmark/labnotes7

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== Photosensor ==
== Photosensor ==
 +
=== Insertion of B0015 in pSB3C5 and pSB3T5 ===
 +
 +
'''Date:''' 8/23 - 8/29 2010<Br>
 +
'''Done By:''' Maria and Lc<Br>
 +
'''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>
 +
==== Pfu PCR amplification and purification of B0015 ====
 +
'''Date:''' 8/23 2010<Br>
 +
'''Done By:''' Maria and Lc<Br>
 +
'''Protocol:'''[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]<br>
 +
'''Notes:'''<br>
 +
4 PCR reactions are prepared. 2uL Miniprep of pSB1AK3 (white 43) are distrubuted in each tube. PCR tubes are marked B00153.A-D.<br>
 +
Premix x5:<br>
 +
<table style="text-align: left;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>pfu buffer + MgSO<small>4</small></td>
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      <td>25uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>dNTP mix</td>
 +
      <td>7.5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>VF2 primer</td>
 +
      <td>7.5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>VR primer</td>
 +
      <td>7.5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>H<small>2</small>0</td>
 +
      <td>190uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>pfu polymerase&nbsp;</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
</table><br><br>
 +
PCR program:<br>
 +
<table style="text-align: left;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>start</td>
 +
      <td>94C</td>
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      <td>3min</td>
 +
    </tr>
 +
    <tr>
 +
      <td>denaturating</td>
 +
      <td>94C</td>
 +
      <td>2min</td>
 +
    </tr>
 +
    <tr>
 +
      <td>annealing</td>
 +
      <td>55C</td>
 +
      <td>30s</td>
 +
    </tr>
 +
    <tr>
 +
      <td>elongation</td>
 +
      <td>72C</td>
 +
      <td>30s</td>
 +
    </tr>
 +
    <tr>
 +
      <td>go to</td>
 +
      <td>2</td>
 +
      <td>rep.29x</td>
 +
    </tr>
 +
    <tr>
 +
      <td>end</td>
 +
      <td>72C</td>
 +
      <td>5min</td>
 +
    </tr>
 +
    <tr>
 +
      <td>hold</td>
 +
      <td>4C</td>
 +
      <td></td>
 +
    </tr>
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</table><br>
 +
5uL of the PCR product was loaded onto a 2% agarose gel. Gene ruler 100bp DNA ladder was used ad marker.<br><br>
 +
'''Results:'''<br>
 +
'''Analysis:'''<br>
 +
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>
 +
End conc.: 48ng/uL <br><br>
 +
 +
==== Restriction digest of B0015 ====
 +
'''Date:''' 8/24 2010<Br>
 +
'''Done By:''' Maria and Lc<Br>
 +
'''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>
 +
'''Notes:'''<br>
 +
Restriction mixture B0015:<br>
 +
<table style="text-align: left;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>H<small>2</small>O</td>
 +
      <td>38uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FD green buffer</td>
 +
      <td>8uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>EcoRI</td>
 +
      <td>4uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PstI</td>
 +
      <td>4uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B0015</td>
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      <td>30uL</td>
 +
    </tr>
 +
</table><br><br>
 +
 +
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>
 +
DNA was extracted from gel according to protocol.<br><br>
 +
'''Results:'''<br>
 +
DNA conc:
 +
<table style="text-align: left;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>sample</td>
 +
      <td>conc. (ng/uL)</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B0015</td>
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      <td>6.5</td>
 +
    </tr>
 +
    <tr>
 +
      <td>pSB3C5</td>
 +
      <td>50.8</td>
 +
    </tr>
 +
    <tr>
 +
      <td>pSB3T5</td>
 +
      <td>9.9</td>
 +
    </tr>
 +
</table><br><br>
 +
'''Analysis:'''
 +
the purified DNA was used for ligation.<br><br>
 +
 +
==== Ligation of PS and pSB1C3 and pCB1AK3 ====
 +
'''Date:''' 8/24 2010<Br>
 +
'''Done By:''' Maria and Lc<Br>
 +
'''Protocol:'''[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.2 LG1.2]<br>
 +
'''Notes:'''<br>
 +
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>
 +
Ligation mixtures (B0015 in pSB3C5):<br>
 +
<table style="text-align: left;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td></td>
 +
      <td>L1</td>
 +
      <td>L2</td>
 +
      <td>L3</td>
 +
    </tr>
 +
    <tr>
 +
      <td>T4 ligase buffer</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>T4 ligase</td>
 +
      <td>1uL</td>
 +
      <td>1uL</td>
 +
      <td>1uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>pSB3C5</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B0015 </td>
 +
      <td>0.7uL</td>
 +
      <td>2uL</td>
 +
      <td>4.5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>H<small>2</small>0</td>
 +
      <td>14.3uL</td>
 +
      <td>13uL</td>
 +
      <td>10.5uL</td>
 +
    </tr>
 +
</table><br>
 +
Ligation mixtures (B0015 in pSB3T5):<br>
 +
<table style="text-align: left;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td></td>
 +
      <td>L1</td>
 +
      <td>L2</td>
 +
      <td>L3</td>
 +
    </tr>
 +
    <tr>
 +
      <td>T4 ligase buffer</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>T4 ligase</td>
 +
      <td>1uL</td>
 +
      <td>1uL</td>
 +
      <td>1uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>pSB3T5</td>
 +
      <td>5uL</td>
 +
      <td>5uL</td>
 +
      <td>5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B0015 </td>
 +
      <td>0.5uL</td>
 +
      <td>1uL</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>H<small>2</small>0</td>
 +
      <td>11.5uL</td>
 +
      <td>11uL</td>
 +
      <td>10uL</td>
 +
    </tr>
 +
</table><br><br>
 +
The samples was incubated at 17C ON at used for transformation<br><br>
 +
 +
==== Transfomation of ligated plasmid in Top 10 E.coli ====
 +
 +
'''Date:''' 8/25 2010<Br>
 +
'''Done By:''' Maria and Lc<Br>
 +
'''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>
 +
'''Notes:'''<br>
 +
The compotent cells and transformation was carried out according to protocol.<br><br>
 +
'''Results:'''<br>
 +
the controle plates were okay, and there were many colonies on plates with cells transformed with either of the ligation mixtures..<br><br>
 +
'''Analysis:'''<br>
 +
The transformation was successfull and colonies was selected and used in coloni PCR.<br>
 +
--[[User:Tipi|Tipi]] 13:41, 26 September 2010 (UTC)<br><br>
 +
=== Colony PCR on B0017 ===
 +
Date: 27/8<br>
 +
Done by: LC<br>
 +
Methods: PCR<br>
 +
Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3 CP1.3]<br>
 +
Notes: <br>
 +
Premix:<br>
 +
12,5 µl 10xTAQ Buffer<br>
 +
5 µl MgCl2 <br>
 +
5 µl VF2 <br>
 +
5 µl VR <br>
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2,5 µl dNTP<br>
 +
17,5 µl H2O<br>
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5/8 µl TAQ Polymerase<br>
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<br>
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9,5 µl Premix were added to 15 µl of H2O containing the lysed cells.<br>
 +
<br>
 +
PCR Program:<br>
 +
<table style="text-align: left;" border="1" cellpadding="2"
 +
cellspacing="2">
 +
<tr>
 +
<td style="vertical-align: top;">Start<br>
 +
</td>
 +
<td style="vertical-align: top;">94&nbsp; C<br>
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</td>
 +
<td style="vertical-align: top;">2 min<br>
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</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Denaturing<br>
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</td>
 +
<td style="vertical-align: top;">94 C<br>
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</td>
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<td style="vertical-align: top;">1 min<br>
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</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Annealing<br>
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</td>
 +
<td style="vertical-align: top;">55 C<br>
 +
</td>
 +
<td style="vertical-align: top;">1 min<br>
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</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Elongation<br>
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</td>
 +
<td style="vertical-align: top;">72 C<br>
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</td>
 +
<td style="vertical-align: top;">30 sec<br>
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</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Goto2<br>
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</td>
 +
<td style="vertical-align: top;">rep<br>
 +
</td>
 +
<td style="vertical-align: top;">29x<br>
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</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">End<br>
 +
</td>
 +
<td style="vertical-align: top;">72 C<br>
 +
</td>
 +
<td style="vertical-align: top;">3 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Hold<br>
 +
</td>
 +
<td style="vertical-align: top;">4 C<br>
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</td>
 +
<td style="vertical-align: top;"><br>
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</td>
 +
</tr>
 +
</table>
 +
<br>
 +
<br>
 +
Results: <br>
 +
[[Image:Team-SDU-Denmark-cPCRB0017.jpg|300px]] <br>
 +
No useable results, only unclear bands around 120 BP, which seem to be the result of mispriming with VR on B0010.
 +
 +
== Retinal ==
==== Futher PCR on POT2 with NinaB (New Primers NO. 5) ====
==== Futher PCR on POT2 with NinaB (New Primers NO. 5) ====
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Done by: Marie & Tommy<br>
Done by: Marie & Tommy<br>
Methods: PCR<br>
Methods: PCR<br>
-
protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]
+
protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]
Notes:<br>
Notes:<br>
NinB2fw and NinaB2rv was used.<br>
NinB2fw and NinaB2rv was used.<br>
Line 25: Line 349:
</head>
</head>
<body>
<body>
-
<table style="text-align: left; width: 100%;" border="1"
+
<table style="text-align: left; width: 300px;" border="1"
  cellpadding="2" cellspacing="2">
  cellpadding="2" cellspacing="2">
     <tr>
     <tr>
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Start date: 24/8<br>  
Start date: 24/8<br>  
Methods: Ligation, Competent cells, Transformation<br>
Methods: Ligation, Competent cells, Transformation<br>
-
Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1], CC1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1], TR1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1]
+
Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1 LG1.1], [https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1 CC1.1], [https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1]
==== DNA purification from PCR ====
==== DNA purification from PCR ====
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</head>
</head>
<body>
<body>
-
<table style="text-align: left; width: 100%;" border="1"
+
<table style="text-align: left; width: 100px;" border="1"
  cellpadding="2" cellspacing="2">
  cellpadding="2" cellspacing="2">
     <tr>
     <tr>
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Done by: Marie & Tommy<br>
Done by: Marie & Tommy<br>
Methods: Ligation<br>
Methods: Ligation<br>
-
protocos:L1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.3]
+
protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.3 L1.3]
Notes:<br>
Notes:<br>
3 ligatons mixtures was made:<br>
3 ligatons mixtures was made:<br>
Line 150: Line 474:
Done by: Marie & Tommy<br>
Done by: Marie & Tommy<br>
Methods: Colony PCR<br>
Methods: Colony PCR<br>
-
protocos:CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]
+
protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]
Notes:<br>
Notes:<br>
15 colonies were picked form different plates, with differnt plasmid to insert ratio: colonie's 1,2,3,13 were picked from plates with 1:1 plasmid to insert ratio, colonie's 4,5,6 were picked from plates with 1:3 plasmid to insert ratio and colonie's 7,8,9,10,11,12,14,15 were picked from plates with 1:6 plasmid to insert ratio.<br>
15 colonies were picked form different plates, with differnt plasmid to insert ratio: colonie's 1,2,3,13 were picked from plates with 1:1 plasmid to insert ratio, colonie's 4,5,6 were picked from plates with 1:3 plasmid to insert ratio and colonie's 7,8,9,10,11,12,14,15 were picked from plates with 1:6 plasmid to insert ratio.<br>
Line 161: Line 485:
</head>
</head>
<body>
<body>
-
<table style="text-align: left; width: 100%;" border="1"
+
<table style="text-align: left; width: 300px;" border="1"
  cellpadding="2" cellspacing="2">
  cellpadding="2" cellspacing="2">
     <tr>
     <tr>
Line 209: Line 533:
Done by: Marie & Tommy<br>
Done by: Marie & Tommy<br>
Methods: Restriction Digest<br>
Methods: Restriction Digest<br>
-
protocos:RD1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1]
+
protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1]
Notes:<br>
Notes:<br>
Restriction digest was performed on tubes 5,6,10 and 11 to test for insertion of ninaB (Correct orientation) XbaI and SPEI was used and a gel was run:<br>
Restriction digest was performed on tubes 5,6,10 and 11 to test for insertion of ninaB (Correct orientation) XbaI and SPEI was used and a gel was run:<br>
Line 217: Line 541:
Done by: Marie & Tommy<br>
Done by: Marie & Tommy<br>
Methods: Colony PCR<br>
Methods: Colony PCR<br>
-
protocos:CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]
+
protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]
Notes:<br>
Notes:<br>
8 new colonies (16-23) were chosen in addition to colonies 5,6,10 and 11 form 25I8-10 colonie PCR.<br>
8 new colonies (16-23) were chosen in addition to colonies 5,6,10 and 11 form 25I8-10 colonie PCR.<br>
Line 228: Line 552:
</head>
</head>
<body>
<body>
-
<table style="text-align: left; width: 100%;" border="1"
+
<table style="text-align: left; width: 300px;" border="1"
  cellpadding="2" cellspacing="2">
  cellpadding="2" cellspacing="2">
     <tr>
     <tr>
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</head>
</head>
<body>
<body>
-
<table style="text-align: left; width: 100%;" border="1"
+
<table style="text-align: left; width: 300px;" border="1"
  cellpadding="2" cellspacing="2">
  cellpadding="2" cellspacing="2">
     <tr>
     <tr>

Latest revision as of 22:31, 23 October 2010