Team:SDU-Denmark/labnotes3

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(Difference between revisions)
(PCR of FlhDC freeye tube 23)
(colony PCR using taq on ligated BBa_J13002 in pSB1C3)
 
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The Concentrations were quite low, but we pooled them (1P+2P and 3B+4B) and used them for ligation.
The Concentrations were quite low, but we pooled them (1P+2P and 3B+4B) and used them for ligation.
<br><br>
<br><br>
 +
 +
==== Ligation of J13002 promotor rbs into pSB3T5 assembly plasmid. ====
 +
Start date: 26/7<br>
 +
Methods: Restriction Digest, Gel Extraction, Ligation, Transformation (not done by me) and colony pcr<br>
 +
Protocols: CP1.3, LG1.3, RD1.1, DE1.3
 +
==== Restriction digest, Gel extracton and ligation of BBa_J13002 and pSB1C3 ====
 +
Date: 27/7<br>
 +
Done by: Christian<br>
 +
Methods: Restriction digest, Gel extraction, ligation and transformation (not by me)<br>
 +
protocos: RD1.1, DE1.3, LG1.3
 +
<br><br>
 +
Restriction digest using EcoRI and PstI and gel extraction were done following protocol. at beginning of ligation concentrations were: 3.7ng/ul part at 95BP and 8ng/ul plasmid at 3215BP. four tubes were run, 1 at 3:1 ration, two at 6:1 and one at 9:1. iGEM consensus protocol was followed, with 20ng vector as starting point. Tubes were cooled for transformations in the morning.
 +
<br><br>
 +
Results: Quite low concentrations were reached, but the iGEM protocol should ensure the same molar ratio and total concentration as was planned for.<br><br>
 +
Analysis: Everything is going according to plan for now.
 +
==== colony PCR using taq on ligated BBa_J13002 in pSB1C3 ====
 +
Date: 28/7<br>
 +
Done by: Christian<br>
 +
Methods: colony pcr<br>
 +
protocos: CP1.3
 +
<br><br>
 +
Colony pcr was run on 8 colonies, and plates were streaked and incubated at 37° ON. protocol was followed to point, with enzyme added to mix. PErhaps too little colony was scraped of original plates, and some tubes were defective, letting out steam during pcr.
 +
<br><br>
 +
Results: A band showed at around 300bp in well 4.1. The expected length was 312bp. wells3.3 and 3.4 show a band outside the blue ladder, that could be rfp (since this was the original insert.) tube 3.2 was ruined.<br>
 +
[[Image:Team-sdu-denmark-Promotor_Rbs_i_psb3t5_colonipcr.jpg | 300px]]
 +
<br><br>
 +
Analysis: Well 3.2 shows promising results. ON and miniprep will be made, and it will be sent for sequencing.<br><br>
=== Pfu PCR of J13002 and B0015 ===
=== Pfu PCR of J13002 and B0015 ===
Line 273: Line 300:
The picture shows no expression of the Double terminator (B0015) and fine expression of Promoter + RBS (J13002). The Double terminator product was not used further, while the promnoter + RBS was run on a gel and extracted (described in notes below. <br><br>  
The picture shows no expression of the Double terminator (B0015) and fine expression of Promoter + RBS (J13002). The Double terminator product was not used further, while the promnoter + RBS was run on a gel and extracted (described in notes below. <br><br>  
--[[User:Louch07|Louch07]] 11:38, 28 July 2010 (UTC)
--[[User:Louch07|Louch07]] 11:38, 28 July 2010 (UTC)
 +
 +
=== Pfu PCR and gel purification of pSB1Ak3 w. B0015 ===
 +
''Done by:'' Pernille <br>
 +
''Date:'' July 28th <br>
 +
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]], [[https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.2 Gel extraction]]<br>
 +
''Notes:'' <br> the PCR was run with the following program:
 +
<html>
 +
<head>
 +
<meta content="text/html; charset=ISO-8859-1"
 +
http-equiv="content-type">
 +
<title></title>
 +
</head>
 +
<body>
 +
<table style="text-align: left; width: 100px;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
<tbody>
 +
<tr>
 +
<td style="vertical-align: top;">Progress<br>
 +
</td>
 +
<td style="vertical-align: top;">Temp. (c elcius)<br>
 +
</td>
 +
<td style="vertical-align: top;">Time (min.)<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Start<br>
 +
</td>
 +
<td style="vertical-align: top;">94<br>
 +
</td>
 +
<td style="vertical-align: top;">3<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Denaturing<br>
 +
</td>
 +
<td style="vertical-align: top;">94<br>
 +
</td>
 +
<td style="vertical-align: top;">2<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Annealing<br>
 +
</td>
 +
<td style="vertical-align: top;">55<br>
 +
</td>
 +
<td style="vertical-align: top;">0.5<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Elongation<br>
 +
</td>
 +
<td style="vertical-align: top;">72<br>
 +
</td>
 +
<td style="vertical-align: top;">0.5<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">GOTO<br>
 +
</td>
 +
<td style="vertical-align: top;"><br>
 +
</td>
 +
<td style="vertical-align: top;">rep x 29<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">End<br>
 +
</td>
 +
<td style="vertical-align: top;">72<br>
 +
</td>
 +
<td style="vertical-align: top;">2<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Hold<br>
 +
</td>
 +
<td style="vertical-align: top;">4<br>
 +
</td>
 +
<td style="vertical-align: top;"><br>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<br>
 +
</body>
 +
</html>
 +
 +
The amplified DNA sequence was run on af 2 agarose gel. the db. terminator are 450bp longe and a visuel band appeared:
=== Pfu PCR of FlhDC with mutation ===
=== Pfu PCR of FlhDC with mutation ===
Line 434: Line 548:
<table style="text-align: left; width: 100px;" border="1"
<table style="text-align: left; width: 100px;" border="1"
cellpadding="2" cellspacing="2">
cellpadding="2" cellspacing="2">
-
<tbody>
 
<tr>
<tr>
<td style="vertical-align: top;">Tube<br>
<td style="vertical-align: top;">Tube<br>
Line 505: Line 618:
</td>
</td>
</tr>
</tr>
-
</tbody>
 
</table>
</table>
<br>
<br>
-
</body>
 
</html>
</html>
<br><br>
<br><br>
Line 520: Line 631:
--[[User:Louch07|Louch07]] 09:06, 29 July 2010 (UTC)
--[[User:Louch07|Louch07]] 09:06, 29 July 2010 (UTC)
-
=== compent cell and transformation of photosensor and promotor+RNA in pSB3T5 ===
+
 
-
<br>
+
 
-
''Done by:'' Maria and Pernille <br>
+
-
''Date:'' July 27th <br>
+
-
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1 CC1.1] and [https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1]<br>
+
-
''Notes:'' <br>
+
-
''Results:'' The OD of the cells were measured every hour and the corelation was:
+
-
<table style="text-align: left; height: 198px; width: 154px;" border="1"
+
-
cellpadding="2" cellspacing="2">
+
-
<tr>
+
-
<td style="vertical-align: top; width: 67px;">Time /h<br>
+
-
</td>
+
-
<td style="vertical-align: top; width: 85px;">OD550 /Abs<br>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td style="vertical-align: top; width: 67px;">1<br>
+
-
</td>
+
-
<td style="vertical-align: top; width: 85px;">0,024<br>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td style="vertical-align: top; width: 67px;">2<br>
+
-
</td>
+
-
<td style="vertical-align: top; width: 85px;">0,048<br>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td style="vertical-align: top; width: 67px;">3<br>
+
-
</td>
+
-
<td style="vertical-align: top; width: 85px;">0,116<br>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td style="vertical-align: top;">4<br>
+
-
</td>
+
-
<td style="vertical-align: top;">0,58<br>
+
-
</td>
+
-
</tr>
+
-
</table>
+
-
<br><br>
+
=== PCR of FlhDC freeze tube 23 ===
=== PCR of FlhDC freeze tube 23 ===
<br>
<br>
Line 568: Line 640:
''Notes:'' Since the PCR of freeze tube 31 shoved pour results on the gel, we are now trying another sample (freeze tube 23) This is made from sample 14 and 16 (mutation PCR samples). The gel picture from these samples showed the strongest band in the samples run with annealing temperature of 64.5 celcius.<br><br>
''Notes:'' Since the PCR of freeze tube 31 shoved pour results on the gel, we are now trying another sample (freeze tube 23) This is made from sample 14 and 16 (mutation PCR samples). The gel picture from these samples showed the strongest band in the samples run with annealing temperature of 64.5 celcius.<br><br>
''Results:'' The PCR result was pooled in freeze tube 49.<br>
''Results:'' The PCR result was pooled in freeze tube 49.<br>
-
[[Image:Team-SDU Denmark Double terminator and flhdc freeze tube 49.jpg]]
+
[[Image:Team-SDU Denmark Double terminator and flhdc freeze tube 49.jpg|300px]]
-
<br> The picture shows a clear band at 200bp and smear. The FlhDC gene is 986bp.
+
<br> The picture shows a clear band at 200bp and smear. The FlhDC gene of 986bp is not seen on the gel. <br>
 +
--[[User:Louch07|Louch07]] 07:26, 5 August 2010 (UTC)
-
== Follow-up colony PCR ===
+
=== Checking if Pst1 site in FlhDC mut is not pressent ===
-
Date: 26/7<br>
+
<br>
-
Methods: Colony PCR<br>
+
''Done by:'' Louise <br>
-
Protocol: CP1.3<br>
+
''Date:'' july 30th <br>
 +
''Protocol:''[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1] <br>
 +
''Notes:'' Two restrictions were made, one with native flhDC containing the Pst1 site (Freeze tube 13) and one with FlhDC mut which should not have a Pst1 site (freeze tube 49). <br>
 +
'''''Restriction mixture:''''' <br>
 +
13 ul water <br>
 +
1 ul Pst1 enzyme <br>
 +
2 ul FD green Buffer <br>
 +
5 ul PCR product <br>
 +
''Results:'' <br>
 +
The result of this Pst1 test was very pour, and is redone later with other samples. <br>
 +
[[Image:Team-SDU Denmark FlhDC mut vs FlhDC native.jpg|300px]]
 +
<br>
 +
--[[User:Louch07|Louch07]] 07:26, 5 August 2010 (UTC)
 +
The gel was 1.5% and a 100-1000 ladder was used.<br><br>
 +
=== Incertion of flhD/C (mutated gene sequence) in pSB3K3 and pSB1C3 ===
 +
<br>
 +
''Done by:'' Maria and Sheila <br><br>
 +
''Parts used:'' K343000 (flhD/C, mutated gene sequence), pSB3K3 and pSB1C3<br><br>
 +
 
 +
==== Digestion  and gelextraction ====
 +
 
 +
''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><br>
 +
''Notes:'' purified flhD/C and miniprep of pSB1C3 and pSB3K3 were digested using the EcoRI and PstI restriction enzymes. <br><br>
 +
Restriction mixture (FlhD/C):<br>
 +
<html><table style="text-align: left; width: 300px;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>H2O</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>FlhD/C DNA (white 48)</td>
 +
      <td>30uL</td>
 +
    </tr>
 +
</table><br>
 +
Restriction mixture (plasmid):<br>
 +
<table style="text-align: left; width: 300px;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td>H2O</td>
 +
      <td>24uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FD green buffer</td>
 +
      <td>4uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>EcoRI</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PstI</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>pSB3K3 or pSB1C3</td>
 +
      <td>10uL</td>
 +
    </tr>
 +
</table></html><br>
 +
The digested samples were loaded onto a 1.5% agarose gel. Undigested FlhD/C and vector was used as controls. Hyperladder II and generuler DNA ladder mix (red) were used as markers.<br>
 +
Digested flhD/C is app. 980bp<br>
 +
pSB3K3 digested with EcoRI and PstI is 2713bp<br>
 +
pSB1C3 digested with EcoRI and PstI is 2035bp<br>
 +
DNA was extracted from gel according to protocol<br><br>
 +
''Results:''<br><br>
 +
 
 +
==== Ligation ====
 +
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.2 LG1.2]<br><br>
 +
''Notes:''<br>
 +
flhD/C is ligated with either pSB1C3 or pSB3K3.<br>
 +
3 ligation mixtures with ratios of 1:3, 1:6 and 1:9 (vector:flhD/C)was prepared.<br>
 +
20-30ng of vector was used for each ligation.<br>
 +
Ligation mixtures (FlhD/C in pSB3K3):<br>
 +
<html><table style="text-align: left; width: 100%;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td></td>
 +
      <td>Lig. 1(1:3)</td>
 +
      <td>Lig. 2 (1:6)</td>
 +
      <td>Lig. 3 (1:9)</td>
 +
    </tr>
 +
    <tr>
 +
      <td>T4 ligase bf.</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>pSB3K3</td>
 +
      <td>2.5uL</td>
 +
      <td>2.5uL</td>
 +
      <td>2.5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FlhD/C</td>
 +
      <td>2uL</td>
 +
      <td>4.5uL</td>
 +
      <td>6.5uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>H20</td>
 +
      <td>12.5uL</td>
 +
      <td>10uL</td>
 +
      <td>8uL</td>
 +
    </tr>
 +
</table></html><br>
 +
Ligation mixtures (flhD/C in pSB1C3):<br>
 +
<table style="text-align: left; width: 100%;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
    <tr>
 +
      <td></td>
 +
      <td>Lig. 1(1:3)</td>
 +
      <td>Lig. 2 (1:6)</td>
 +
      <td>Lig. 3 (1:9)</td>
 +
    </tr>
 +
    <tr>
 +
      <td>T4 ligase bf.</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>pSB1C3</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
      <td>2uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FlhD/C</td>
 +
      <td>4uL</td>
 +
      <td>5uL</td>
 +
      <td>6uL</td>
 +
    </tr>
 +
    <tr>
 +
      <td>H20</td>
 +
      <td>11uL</td>
 +
      <td>10uL</td>
 +
      <td>9uL</td>
 +
    </tr>
 +
</table><br>
 +
All samples were incubated at 17 degrees until they were used for transformation.<br><br>
 +
 
 +
=== Follow-up colony PCR ===
 +
''Date:'' 26/7<br>
 +
''Methods:'' Colony PCR<br>
 +
''Protocol:'' CP1.3<br>
Experiment done by: Maria, LC  
Experiment done by: Maria, LC  
<br><br>
<br><br>
-
Notes: We made a sample for every plate from the last colony PCR. Out of the 29 samples we could only run 25 at once in the PCR machine. Plate 23 was missing, so there is no sample for it.<br><br>
+
''Notes:'' We made a sample for every plate from the last colony PCR. Out of the 29 samples we could only run 25 at once in the PCR machine. Plate 23 was missing, so there is no sample for it.<br><br>
-
Results: [[Image:Team-SDU-Denmark-colpcr267.jpg|600px]]<br>
+
''Results:'' <br>
 +
[[Image:Team-SDU-Denmark-colpcr267.jpg|300px]]<br>
Lengths of PCR products: <br>
Lengths of PCR products: <br>
1200: 4, 5, 6, 9, 10, 14, 21, 26<br>
1200: 4, 5, 6, 9, 10, 14, 21, 26<br>
Line 589: Line 832:
Colonies 1, 15, 16 and 22 gave no result.
Colonies 1, 15, 16 and 22 gave no result.
<br><br>
<br><br>
-
Analysis: Since we had so many different lengths, we will cut one from each length, specifically colony: 2, 8, 11, 13, 20, 24, 26.
+
''Analysis:'' Since we had so many different lengths, we will cut one from each length, specifically colony: 2, 8, 11, 13, 20, 24, 26.
<br><br>
<br><br>
Line 911: Line 1,154:
''Results:''<br>
''Results:''<br>
gel electrophoresis:<br>
gel electrophoresis:<br>
-
[[Image:Team-SDU-Denmark-Digestwithxandp.jpg|600px]]<br><br>
+
[[Image:Team-SDU-Denmark-Digestwithxandp.jpg|300px]]<br><br>
''Analysis:''<br>
''Analysis:''<br>
The lanes containing digested PCR product from coloni #5, 6 and 21 have a band smear around 100-200bp,and a band at around 800bp.<br>
The lanes containing digested PCR product from coloni #5, 6 and 21 have a band smear around 100-200bp,and a band at around 800bp.<br>
Line 958: Line 1,201:
''Results:''<br>
''Results:''<br>
Gel electrophoresis:<br>
Gel electrophoresis:<br>
-
[[Image:Team-SDU-Denmark-Miniprepcoloni5,6,21.jpg|600px]]<br><br>
+
[[Image:Team-SDU-Denmark-Miniprepcoloni5,6,21.jpg|300px]]<br><br>
''Analysis:''<br>
''Analysis:''<br>
we have purified the plasmids.The concentrations are high but not as high as expected when the cells are boosted prior to the miniprep.<br> In order to obtain an even higher concentration transfer all 5mL ON culture to 20mL pre heated LB media. run all 25mL new culture as 1 miniprep.<br> Due to the low concentrations we need to dry down our samples before they are ready for sequentation.<br>
we have purified the plasmids.The concentrations are high but not as high as expected when the cells are boosted prior to the miniprep.<br> In order to obtain an even higher concentration transfer all 5mL ON culture to 20mL pre heated LB media. run all 25mL new culture as 1 miniprep.<br> Due to the low concentrations we need to dry down our samples before they are ready for sequentation.<br>
Line 969: Line 1,212:
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1]] and gelpurificaton (DE1.3)<br><br>
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1]] and gelpurificaton (DE1.3)<br><br>
''Notes:''because the concentration for both plasmid and biobrick is rather small I dobbelt the volumen of the reagent compared to the protocol. in the soultion contaning the brick the volumen of the PCR product was triple and to adjust the total volumen i added 5ul less water.  We made 2 Restriction mixtures which both were 4 times the mixture in the protocol. both the plasmid and brick was cut at the E and P site. After the cutting the plasmid and brick was run on seperate gels because of there different size. the plasmid on a 1,5% gel and brick on a 2% agarose gel. When I observed the gel under the UV lamp the was no visible band on the gel were the brick was loaded. therefore it was only possible to do DNA extrated from the gel contraining the plasmid. The Dna was eluted in 10ul of water and I eluted two times. The DNA concentration was measured on the NaonDrop and was found to 55,41ng/ul for the first elution and 28,03ng/ul for the second. the to samples are pooled and stored in a freeze tube.<br><br>
''Notes:''because the concentration for both plasmid and biobrick is rather small I dobbelt the volumen of the reagent compared to the protocol. in the soultion contaning the brick the volumen of the PCR product was triple and to adjust the total volumen i added 5ul less water.  We made 2 Restriction mixtures which both were 4 times the mixture in the protocol. both the plasmid and brick was cut at the E and P site. After the cutting the plasmid and brick was run on seperate gels because of there different size. the plasmid on a 1,5% gel and brick on a 2% agarose gel. When I observed the gel under the UV lamp the was no visible band on the gel were the brick was loaded. therefore it was only possible to do DNA extrated from the gel contraining the plasmid. The Dna was eluted in 10ul of water and I eluted two times. The DNA concentration was measured on the NaonDrop and was found to 55,41ng/ul for the first elution and 28,03ng/ul for the second. the to samples are pooled and stored in a freeze tube.<br><br>
 +
== Group: Retinal ==
== Group: Retinal ==
=== Colony PCR and extraction of ninaB gene from transformants ===
=== Colony PCR and extraction of ninaB gene from transformants ===
Line 983: Line 1,227:
<br><br>
<br><br>
Results:_Expected length at 1920 BP did not show. In fact no bands showed, except somethin like a primer smear around 100bp.<br>
Results:_Expected length at 1920 BP did not show. In fact no bands showed, except somethin like a primer smear around 100bp.<br>
-
[[Image:Team-SDU-Denmark-NinaB taq colony pcr on transformants.jpg | 400px ]]
+
[[Image:Team-SDU-Denmark-NinaB taq colony pcr on transformants.jpg | 300px ]]
<br><br>
<br><br>
Analysis: Anealing temperature might have been set to high, as the primers were constructed to aneal at 55°. another run will be done on miniprepped plasmids, with a different program.
Analysis: Anealing temperature might have been set to high, as the primers were constructed to aneal at 55°. another run will be done on miniprepped plasmids, with a different program.
 +
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI ====
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI ====
Date: 27/7<br>
Date: 27/7<br>
Line 995: Line 1,240:
<br><br>
<br><br>
Results: strange things going on at different lengths, and nothing matches the expected length of about 3800BP.<br>
Results: strange things going on at different lengths, and nothing matches the expected length of about 3800BP.<br>
-
[[Image:Team-SDU-Denmark-NinaB miniprep and first restriction digest-1.jpg | 800px]]
+
[[Image:Team-SDU-Denmark-NinaB miniprep and first restriction digest-1.jpg |500px]]
<br>
<br>
Analysis: Another digest is needed to show that plasmid length is wrong, as it seems unlikely with comercial plasmids.<br>
Analysis: Another digest is needed to show that plasmid length is wrong, as it seems unlikely with comercial plasmids.<br>
 +
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
Date: 28/7<br>
Date: 28/7<br>
Line 1,007: Line 1,253:
<br><br>
<br><br>
results: Digest showed a band close to the 3.8kb marker expected for the full length plasmid, concurrent with only one digest working. (although an earlier digest with EcoRI alone showed a different band.) Miniprepped plasmid had also "moved up" the ladder<br>
results: Digest showed a band close to the 3.8kb marker expected for the full length plasmid, concurrent with only one digest working. (although an earlier digest with EcoRI alone showed a different band.) Miniprepped plasmid had also "moved up" the ladder<br>
-
[[Image:Team-SDU-Denmark-NinaB restriction digest with eco and xho-1.jpg | 400px ]]
+
[[Image:Team-SDU-Denmark-NinaB restriction digest with eco and xho-1.jpg | 300px ]]
<br><br>
<br><br>
analysis: Something fishy is up with this plasmid, and the digest. one more will be run only using XhoI.
analysis: Something fishy is up with this plasmid, and the digest. one more will be run only using XhoI.
 +
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
Date: 28/7<br>
Date: 28/7<br>
Line 1,019: Line 1,266:
<br><br>
<br><br>
results: Gel showed a band between 3.5K and 4k markers, consistent with earlier results, indicating that the plasmid and insert have correct length. Strangely the cut segment has traveled further through the gell than the un-cut control.<br>
results: Gel showed a band between 3.5K and 4k markers, consistent with earlier results, indicating that the plasmid and insert have correct length. Strangely the cut segment has traveled further through the gell than the un-cut control.<br>
-
[[Image:Team-SDU-Denmark-NinaB restriction digest with xhoI.jpg | 400px ]]
+
[[Image:Team-SDU-Denmark-NinaB restriction digest with xhoI.jpg | 300px ]]
<br><br>
<br><br>
-
analysis: There realy is something fishy going on, or as we say in Denmark, there are owls in the marsh. At least the plasmid seems correct.<br>
+
analysis: There realy is something fishy going on, or as we say in Denmark, there are owls in the marsh. At least the plasmid seems correct.<br><br>
-
=== Ligation of J13002 promotor rbs into pSB3T5 assembly plasmid. ===
+
 
-
Start date: 26/7<br>
+
==== PCR on RD products from above experiments ====
-
Methods: Restriction Digest, Gel Extraction, Ligation, Transformation (not done by me) and colony pcr<br>
+
Date: 29/7<br>
-
Protocols: CP1.3, LG1.3, RD1.1, DE1.3
+
-
==== Restriction digest, Gel extracton and ligation of BBa_J13002 and pSB1C3 ====
+
-
Date: 27/7<br>
+
Done by: Christian<br>
Done by: Christian<br>
-
Methods: Restriction digest, Gel extraction, ligation and transformation (not by me)<br>
+
Methods: Gradient taq pcr<br>
-
protocos: RD1.1, DE1.3, LG1.3
+
protocos: CP1.3 (using gradient program)
<br><br>
<br><br>
-
Restriction digest using EcoRI and PstI and gel extraction were done following protocol. at beginning of ligation concentrations were: 3.7ng/ul part at 95BP and 8ng/ul plasmid at 3215BP. four tubes were run, 1 at 3:1 ration, two at 6:1 and one at 9:1. iGEM consensus protocol was followed, with 20ng vector as starting point. Tubes were cooled for transformations in the morning.
+
Notes: 7 small eppendorff tubes were loaded of each product. Protocol for 25ul taq reaction was followed, with out changes other than the gradient programme. One tube (XE7) was tipped and might have spilled a bit of the mix.
 +
The gradient temperatures were the same for each corresponding tube in the three series: E cut with ecoRI, XE cut with both, and X cut with XhoI. Temperatures were from 45-65 degrees. The rest of the program was identical to protocol, with a 2 minute runtime. Custom ninaB primers were used.
<br><br>
<br><br>
-
Results: Quite low concentrations were reached, but the iGEM protocol should ensure the same molar ratio and total concentration as was planned for.<br><br>
+
Results: PCR products were run on a 1% agarose gel, with hyperladder II. A band was expected at 1920BP. A weak bands showed at the correct location in almost all wells (Except the spilled tube). Smears showed in E2 and 3. The clearest bands were at temperatures around 60-65 degrees.
-
Analysis: Everything is going according to plan for now.
+
-
==== colony PCR using taq on ligated BBa_J13002 in pSB1C3 ====
+
-
Date: 28/7<br>
+
-
Done by: Christian<br>
+
-
Methods: colony pcr<br>
+
-
protocos: CP1.3
+
<br><br>
<br><br>
-
Colony pcr was run on 8 colonies, and plates were streaked and incubated at 37° ON. protocol was followed to point, with enzyme added to mix. PErhaps too little colony was scraped of original plates, and some tubes were defective, letting out steam during pcr.
+
We have succesfully shown that our primers will generate a band at our expected length, albeit a very weak one. Perhaps it takes some luck for the reaction to get started. We hope to repeat the experiment with pfu, to get a good product for ligation. We don't know if the cutting has made the difference between this round of pcr and the one performed a couple of days earlier, or if other factors, like the gradient have played in.
-
<br><br>
+
 
-
Results: A band showed at around 300bp in well 4.1. The expected length was 312bp. wells3.3 and 3.4 show a band outside the blue ladder, that could be rfp (since this was the original insert.) tube 3.2 was ruined.<br>
+
 
-
[[Image:Team-sdu-denmark-Promotor_Rbs_i_psb3t5_colonipcr.jpg | 400px]]
+
-
<br><br>
+
-
Analysis: Well 3.2 shows promising results. ON and miniprep will be made, and it will be sent for sequencing.<br><br>
+
== Group: Photosensor ==
== Group: Photosensor ==
Line 1,055: Line 1,291:
''Done by:'' Maria<br>
''Done by:'' Maria<br>
''Methods:'' pfu PCR, gel electrophoresis<br>
''Methods:'' pfu PCR, gel electrophoresis<br>
-
protocos: [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]<br><br>
+
protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]<br><br>
''Notes:''<br>
''Notes:''<br>
gel purified PCR product of BOO15 and J12003 (25 and 26 white) was used as template.<br>
gel purified PCR product of BOO15 and J12003 (25 and 26 white) was used as template.<br>
Line 1,107: Line 1,343:
pfu program:<br>
pfu program:<br>
<html>
<html>
-
<head>
 
-
<meta content="text/html; charset=ISO-8859-1"
 
-
http-equiv="content-type">
 
-
<title></title>
 
-
</head>
 
-
<body>
 
<table style="text-align: left; width: 100px;" border="1"
<table style="text-align: left; width: 100px;" border="1"
cellpadding="2" cellspacing="2">
cellpadding="2" cellspacing="2">
Line 1,174: Line 1,404:
</tbody>
</tbody>
</table>
</table>
-
<br>
 
-
</body>
 
</html>
</html>
<br>
<br>
Line 1,206: Line 1,434:
</td>
</td>
</tr>
</tr>
-
</table><html><br><br>
+
</table></html><br><br>
''Results:''<br>
''Results:''<br>
gel electrophoresis:<br>
gel electrophoresis:<br>
-
[[Image:Team-SDU-Denmark-Amp.ofJ13002andB0015.jpg|600px]]<br><br>
+
[[Image:Team-SDU-Denmark-Amp.ofJ13002andB0015.jpg|300px]]<br><br>
''Analysis:''<br>
''Analysis:''<br>
The PCR product is OK and is used as template for pfu PCR.<br>
The PCR product is OK and is used as template for pfu PCR.<br>
Line 1,216: Line 1,444:
BOO15 is stored as 43 (white)<br>
BOO15 is stored as 43 (white)<br>
--[[User:Tipi|Tipi]] 06:52, 29 July 2010 (UTC)<br><br>
--[[User:Tipi|Tipi]] 06:52, 29 July 2010 (UTC)<br><br>
 +
 +
=== compent cell and transformation of photosensor and promotor+RNA in pSB3T5 ===
 +
<br>
 +
''Done by:'' Maria and Pernille <br>
 +
''Date:'' July 27th <br>
 +
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1 CC1.1] and [https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1]<br>
 +
''Notes:'' <br>
 +
''Results:'' The OD of the cells were measured every hour and the corelation was:
 +
<table style="text-align: left; height: 198px; width: 154px;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
<tr>
 +
<td style="vertical-align: top; width: 67px;">Time /h<br>
 +
</td>
 +
<td style="vertical-align: top; width: 85px;">OD550 /Abs<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 67px;">1<br>
 +
</td>
 +
<td style="vertical-align: top; width: 85px;">0,024<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 67px;">2<br>
 +
</td>
 +
<td style="vertical-align: top; width: 85px;">0,048<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 67px;">3<br>
 +
</td>
 +
<td style="vertical-align: top; width: 85px;">0,116<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">4<br>
 +
</td>
 +
<td style="vertical-align: top;">0,58<br>
 +
</td>
 +
</tr>
 +
</table>
 +
<br><br>
=== Coloni PCR of photosensor in pUC19 from transformation 27/7 ===
=== Coloni PCR of photosensor in pUC19 from transformation 27/7 ===
Line 1,337: Line 1,607:
The PCR samples was loaded onto a 1% agarose gel. Gene ruler DNA ladder mix (red) was used as marker.<br><br>
The PCR samples was loaded onto a 1% agarose gel. Gene ruler DNA ladder mix (red) was used as marker.<br><br>
''Results:''<br>
''Results:''<br>
-
[[Image:Team-SDU-Denmark-coloniPCRPS.jpg|600px]]<br><br>
+
[[Image:Team-SDU-Denmark-coloniPCRPS.jpg|300px]]<br><br>
''Analysis:''<br>
''Analysis:''<br>
No bands are seen in any of the samples. This could be because the VF2 and VR primers does not anneal to the plasmid.<br>
No bands are seen in any of the samples. This could be because the VF2 and VR primers does not anneal to the plasmid.<br>
Line 1,461: Line 1,731:
</table><br>
</table><br>
PCR samples are loaded onto a 1% agarose gel. Gene ruler DNA ladder mix (red) is used as marker.<br><br>
PCR samples are loaded onto a 1% agarose gel. Gene ruler DNA ladder mix (red) is used as marker.<br><br>
-
''Results:''<br><br>
+
''Results:''<br>
 +
[[Image:Team-SDU-Denmark-PSplasmid PCR.jpg|300px]]<br><br>
 +
''Analysis:''<br>
 +
Two weak bands at app. 4500bp and 850bp respectively. The upper band could correspond to the pKJ606 plasmid with insert, and the lower band might indicate VF2-VR without insert, rendering these results rather inconlclusive. Therefore an ON culture for miniprep was made. <br>
 +
--[[User:Lclund|Lclund]] 06:47, 4 August 2010 (UTC)<br><br>
==== Restriction digest and PCR on ninaB plasmids ====
==== Restriction digest and PCR on ninaB plasmids ====
<br>
<br>
Line 1,474: Line 1,748:
3: pOT2 with ''ninaB'' cut with EcoRI and XhoI (EX)<br>
3: pOT2 with ''ninaB'' cut with EcoRI and XhoI (EX)<br>
A 1.5% agarose gel was run:<br>
A 1.5% agarose gel was run:<br>
-
[[Image:team-sdu-denmark-NinaB_restriction_E.jpg | 400px]]
+
[[Image:team-sdu-denmark-NinaB_restriction_E.jpg | 300px]]
<br>
<br>
Nucleic acid concentrations were measured with NanoDrop after gel extraction and purification:  
Nucleic acid concentrations were measured with NanoDrop after gel extraction and purification:  

Latest revision as of 21:27, 23 October 2010