Team:Calgary/26 May 2009

From 2009.igem.org

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<div class="heading">NOTEBOOK PAGE INDEX</div>
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<div class="heading">THIS MONTH</div>
<div class="desc">
<div class="desc">
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<a href="#Carol">Carol</a><br>
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<center>
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<a href="#Emily">Emily</a><br>
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<a href="#Jamie">Jamie</a><br>
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|{{#calendar: query=preload=Team:Calgary/NotebookPreload | year=2009 | month=05 | title=Team:Calgary}}
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<a href="#Kevin">Kevin</a><br>
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|}
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<a href="#Vicki">Vicki</a><br>
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<br>
<br>
<div class="heading">
<div class="heading">
-
Descriptive title of what you're doing - no need for caps
+
Plasmid Isolation of ''luxCDABE'' in TOPO vector
</div>
</div>
<br>
<br>
<div class="desc">
<div class="desc">
</html>
</html>
-
WIKI CODING HERE
+
The purpose of plasmid isolation is to extract plasmids from ''E. coli'' that has topo vector with ''luxCDABE''. Overnight cultures were prepared and Sigma Mini Prep kit was used to to isolate plasmids (see protocols for detailed procedures). The plasmids was finally eluted in the elution buffer provided by the kit to a total of 50uL.
 +
 
 +
The concentration of plasmids was determined using the nano-drop apparatus at 260nm wavelength (see protocols for detailed procedures) and the following table summarizes the concentrations of plasmids.
 +
 
 +
<br>
 +
<br>
 +
<center>
 +
{| border="1"
 +
| '''Plasmid''' || '''260/280'''    || '''260/230''' || '''Concentration [ng/μL]'''
 +
|-
 +
| ''luxCDABE'' TOPO C1  || 2.21 || 4.58 || 47.6
 +
|-
 +
| ''luxCDABE'' TOPO C2|| 2.09 ||  3.39 || 56.0
 +
|}
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*Protocol: Used Sigma GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions.
+
*Objective: To extract plasmids of LuxOD47E in pCR2.1-TOPO vector from overnight cultures.
-
<br>
+
 
-
<br>
+
*Protocol: Used Sigma GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH20.
*Results: Used Nanodrop 1000 Spectrophotometer read at 260 wavelength to determine DNA concentrations
*Results: Used Nanodrop 1000 Spectrophotometer read at 260 wavelength to determine DNA concentrations
-
<br>
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-
<br>
+
<center>
{| border="1"
{| border="1"
| '''Plasmid''' || '''260/280'''    || '''260/230''' || '''Concentration [ng/μL]'''
| '''Plasmid''' || '''260/280'''    || '''260/230''' || '''Concentration [ng/μL]'''
|-
|-
-
| LuxOD47E TOPO C1  || 1.88 || 2.21 || 409.5
+
| ''LuxO''D47E TOPO C1  || 1.88 || 2.21 || 409.5
|-
|-
-
| LuxOD47E TOPO C2|| 1.86 ||  2.18 ||551.4
+
| ''LuxO''D47E TOPO C2|| 1.86 ||  2.18 ||551.4
|-
|-
-
| LuxOD47E BBK C1|| 2.04|| 5.01|| 551.4
+
| ''LuxO''D47E BBK C1|| 2.04|| 5.01|| 551.4
|-
|-
-
| LuxOD47E BBK C2|| 1.77|| 1.39|| 92.9
+
| ''LuxO''D47E BBK C2|| 1.77|| 1.39|| 92.9
|}
|}
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Sigma Genelute Plasmid Mini-prep kit (lot # 048k6062)as used. Standard manufacturer protocol used; elution in 50&#181;L of ddH<sub>2</sub>O.
Sigma Genelute Plasmid Mini-prep kit (lot # 048k6062)as used. Standard manufacturer protocol used; elution in 50&#181;L of ddH<sub>2</sub>O.
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<td width="100px"><u>Plasmid sample</u></td>
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<td width="50px"><u>260/280</u></td>
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<td width="50px"><u>260/230</u></td>
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<td width="200px"><u>Concentration(ng/&#181;L)</u></td>
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<tr style="border-bottom:1px solid #fff; border-top:1px solid #fff;">
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<td width="100px">Plasmid sample</td>
 +
<td width="50px">260/280</td>
 +
<td width="50px">260/230</td>
 +
<td width="150px">Concentration(ng/&#181;L)</td>
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<br>
<br>
<div class="heading">
<div class="heading">
-
Descriptive title of what you're doing - no need for caps
+
Plasmid Isolation of luxOD47A in pCR2.1 TOPO and psB1AC3 vectors
</div>
</div>
<br>
<br>
<div class="desc">
<div class="desc">
</html>
</html>
-
WIKI CODING HERE
+
The purpose was to isolate plasmid containing luxOD47A, as a means of starting the construction of the mutant circuits. TOPO T/A and psB1AC3 vectors containing luxOD47A were isolated using the Sigma GenElute Plasmid Mini-prep kit. 100μL of ddH2O were used to elute, and the purity and concentration of each plasmid were measured using the NanoDrop Spectrophotometer.
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 +
<html>
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<tr>
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<td>
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<a name="Katie"></a>
 +
<br>
 +
<div class="heading">
 +
KATIE
 +
</div>
 +
<br>
 +
<div class="heading">
 +
Challenges Faced with PCR
 +
</div>
 +
<br>
 +
<div class="desc">
 +
</html>
 +
* Working on the virtual PCR machine and completed a general PCR first
 +
* Making it work for PCR with three different types of DNA and placed it initially in three separate scripts and only one would run if it had all of its items. I really want to find a better way to do this so I will continue to work on this.
 +
 
 +
Problem: Items are not being found and it is causing an error on the debug channel and it was not happening before so I will have to find what I have changed in the past day to see what went wrong. It seems to be something that goes wrong within the for loop I have since it seems to be locating everything when there are items missing. I will most likely see to this problem tomorrow.
<html>
<html>
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<br>
<br>
<div class="heading">
<div class="heading">
-
Descriptive title of what you're doing - no need for caps
+
Plasmid Isolation of <i>Pqrr4</i> for Reporter circuit
</div>
</div>
<br>
<br>
<div class="desc">
<div class="desc">
 +
The <i>Pqrr4</i> was isolated from TOPO T/A and biobrick AC (pSB1AC3) using Sigma’s Genelute Plasmid Mini-prep kit (sigma) according to the specifications of the manufacturer. Nanodrop utility along with the Spectrophotometer was used to measure the concentration and purity of the isolated plasmid. The wavelength was set at 260nm.
 +
<br>
 +
<br>
 +
<center>
 +
<b>HTML Code, just as an example for now</b>
 +
<table border="1">
 +
<tr>
 +
<td> <b>Plasmid</b> </td><td> <b>260/280</b>    </td><td> <b>260/230</b> </td><td> <b>Concentration [ng/μL]</b>
 +
</td></tr>
 +
<tr>
 +
<td> <i>Pqrr4</i> TOPO Blue tube </td><td><center> 1.81 </center></td><td><center> 2.24 </center></td><td> 181
 +
</td></tr>
 +
<tr>
 +
<td> <i>Pqrr4</i> TOPO Pink tube </td><td><center> 1.81 </center></td><td><center> 2.02 </center></td><td> 231.3
 +
</td></tr>
 +
<tr>
 +
<td> <i>Pqrr4</i> BBK Green tube </td><td><center> 1.76 </center></td><td><center> 1.66 </center></td><td> 51.2
 +
</td></tr>
 +
<tr>
 +
<td> <i>Pqrr4</i> BBK Pink tube </td><td> 1.85 </td><td> 1.45 </td><td> 60.6
 +
</td></tr></table></center>
 +
<br>
 +
*The two TOPO vectors were from the same colony, thus labelled "Green tube" instead of C1. The two BBK plasmids were also from same colony, so same label applies.
</html>
</html>
-
WIKI CODING HERE
+
<center>
 +
<br>
 +
<b>Wiki Code</b> <br>
 +
Table 1. Purity and concentration of <i>Pqrr4</i> measured with Nanodrop utility and Spectrophotometer
 +
{| border="1"
 +
| '''Plasmid''' || '''260/280'''    || '''260/230''' || '''Concentration [ng/μL]'''
 +
|-
 +
| ''Pqrr4'' TOPO Blue Tube|| 1.81 || 2.24 || 181
 +
|-
 +
| ''Pqrr4'' TOPO Pink Tube|| 1.81 || 2.02 ||231.3
 +
|-
 +
| ''Pqrr4'' BBK Green Tube|| 1.76 || 1.66 || 51.2
 +
|-
 +
| ''Pqrr4'' BBK Pink Tube|| 1.85 || 1.45 || 60.6
 +
|}
 +
</center>
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<html>
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<a name="Patrick"></a>
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<br>
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<div class="heading">
 +
PATRICK
 +
</div>
 +
<br>
 +
<div class="heading">
 +
What's in a (DNA Part) Name?
 +
</div>
 +
<br>
 +
<div class="desc">
 +
</html>
 +
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
 +
 +
Continued developing the script for sending names from one part to the next, so that each could learn where the other was. The problem was that each DNA part in a polymer needs to know the key of the next part, in order to direct the RNA Polymerase to the next part when it visits the current one. Yet object keys weren't stable between sessions, so we needed some additional internal name for each part to go by. Each part would know its internal name, and the name of the next part in the sequence. Then it can use that name to obtain the key of the next part via a message and reply.
 +
 +
I first began to use comma separated values (CSV) to encode my messages, which worked so well I never needed to change it. This is thanks to some good built in tools for CSV handling in the linden scripting language.
 +
 +
<html>
 +
</div>
 +
</td>
 +
</tr>
 +
 +
<tr>
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<td height="10px" bgcolor="#222222">
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 +
<tr>
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 +
<a name="Prima"></a>
 +
<br>
 +
<div class="heading">
 +
PRIMA
 +
</div>
 +
<br>
 +
<div class="heading">
 +
Back from Dragon's Den
 +
</div>
 +
<br>
 +
<div class="desc">
 +
</html>
 +
We returned home on the evening of May 26th, weary from the experience and yet somehow craving more of it. Having satisfied both of our original ambitions, we were pleased with the result and thankful that we were more than just another meal for a few Dragons in the vicinity. Armed with a new sense of promotional direction, more contacts who can help support our initiatives, and the same stunning good looks that we had before we left, we are excited to implement some of these ideas to help capitalize on our marketing potential.
 +
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</td>
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<br>
<br>
<div class="heading">
<div class="heading">
-
Descriptive title of what you're doing - no need for caps
+
Plasmid isolation with mini-prep
</div>
</div>
<br>
<br>
<div class="desc">
<div class="desc">
</html>
</html>
-
WIKI CODING HERE
+
 
 +
<b>Purpose:</b><br>
 +
To isolate the plasmid DNA from the rest of the bacteria for colonies containing LuxPQ on TOPO II Blunt and LuxPQ BBk on psB1AC3.
 +
 
 +
<br>
 +
Reference: The user manual and the Sigma Aldrich site
 +
 
 +
<br>
 +
Kit: MP70 GeneElute Plasmid mini-prep kit. Note that it had been previously used, which may be a problem for contamination.
 +
 
 +
<br>
 +
<b>Materials and methods:</b><br>
 +
Procedure was carried out in accordance with our Plasmid Isolation protocol. When complete, we stored our isolated plasmids in the freezer for concentration measurement and content analysis over the next 2 days.
 +
 
 +
<br>
 +
<b>Results:</b><br>
 +
We were left with tubes containing the described plasmids. Quantifiable details on their concentrations and actual contents are outlined on the May 27 and May 28 entries.
 +
 
 +
<html>
 +
<br>
 +
<div class="heading">
 +
DNA concentration measurements with the Nanodrop spectrophotometer
 +
</div>
 +
<br>
 +
<div class="desc">
 +
</html>
 +
 
 +
<b>Purpose:</b><br> To measure the DNA concentration of LuxPQ plasmids that were isolated in the May 26 miniprep
 +
 
 +
<br>
 +
<b>Materials and Methods:</b><br> The DNA measurement area was cleaned with isopropanol and KimWipes to clear off any trace residue from previous uses. The sample wavelength was set to 260 nm. A blank measurement was made with 1 uL of ddHOH. 1 uL of the sample in question was added to the Nanodrop measurement area. The measurement was made; the sample area was wiped down; and a new sample was measured.
 +
 
 +
<br>
 +
<b>Results:</b>
 +
<br>
 +
<br>
 +
<center>
 +
{| border="1"
 +
| '''Plasmid''' || '''260/280'''    || '''260/230''' || '''Concentration [ng/μL]'''
 +
|-
 +
| ''LuxPQ'' TOPO C1  || 1.83 || 1.86 || 338.2
 +
|-
 +
| ''LuxPQ'' TOPO C2|| 1.81 || 1.87 || 284.2
 +
|-
 +
| ''LuxPQ'' BBK C1|| 1.90 || 2.34 || 295.5
 +
|-
 +
| ''LuxPQ'' BBK C2|| 1.90 || 2.29 || 313.2
 +
|}
 +
</center>
<html>
<html>

Latest revision as of 02:54, 19 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31


NOTEBOOK PAGE INDEX
Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki



CALENDAR

May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31


June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30


July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31


August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31


September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30


October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31



MAY 26, 2009


CAROL

Plasmid Isolation of ''luxCDABE'' in TOPO vector

The purpose of plasmid isolation is to extract plasmids from E. coli that has topo vector with luxCDABE. Overnight cultures were prepared and Sigma Mini Prep kit was used to to isolate plasmids (see protocols for detailed procedures). The plasmids was finally eluted in the elution buffer provided by the kit to a total of 50uL.

The concentration of plasmids was determined using the nano-drop apparatus at 260nm wavelength (see protocols for detailed procedures) and the following table summarizes the concentrations of plasmids.



Plasmid 260/280 260/230 Concentration [ng/μL]
luxCDABE TOPO C1 2.21 4.58 47.6
luxCDABE TOPO C2 2.09 3.39 56.0


EMILY

Plasmid Isolation of LuxOD47E in pCR2.1 TOPO and psB1AC3 vectors

  • Objective: To extract plasmids of LuxOD47E in pCR2.1-TOPO vector from overnight cultures.
  • Protocol: Used Sigma GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH20.
  • Results: Used Nanodrop 1000 Spectrophotometer read at 260 wavelength to determine DNA concentrations
Plasmid 260/280 260/230 Concentration [ng/μL]
LuxOD47E TOPO C1 1.88 2.21 409.5
LuxOD47E TOPO C2 1.86 2.18 551.4
LuxOD47E BBK C1 2.04 5.01 551.4
LuxOD47E BBK C2 1.77 1.39 92.9


JAMIE

Plasmid Isolation of luxOU in pCR-Blunt II-TOPO and psB1AC3

Sigma Genelute Plasmid Mini-prep kit (lot # 048k6062)as used. Standard manufacturer protocol used; elution in 50µL of ddH2O.

Plasmid sample 260/280 260/230 Concentration(ng/µL)
TOP10 luxOU C1 1.91 2.58 365.6
TOP10 luxOU C2 1.90 2.52 287.2
BBK luxOU C1 1.87 2.22 534.2
BBK luxOU C2 1.92 2.24 715.2


JEREMY

Plasmid Isolation of luxOD47A in pCR2.1 TOPO and psB1AC3 vectors

The purpose was to isolate plasmid containing luxOD47A, as a means of starting the construction of the mutant circuits. TOPO T/A and psB1AC3 vectors containing luxOD47A were isolated using the Sigma GenElute Plasmid Mini-prep kit. 100μL of ddH2O were used to elute, and the purity and concentration of each plasmid were measured using the NanoDrop Spectrophotometer.


KATIE

Challenges Faced with PCR

  • Working on the virtual PCR machine and completed a general PCR first
  • Making it work for PCR with three different types of DNA and placed it initially in three separate scripts and only one would run if it had all of its items. I really want to find a better way to do this so I will continue to work on this.

Problem: Items are not being found and it is causing an error on the debug channel and it was not happening before so I will have to find what I have changed in the past day to see what went wrong. It seems to be something that goes wrong within the for loop I have since it seems to be locating everything when there are items missing. I will most likely see to this problem tomorrow.


KEVIN

Plasmid Isolation of Pqrr4 for Reporter circuit

The Pqrr4 was isolated from TOPO T/A and biobrick AC (pSB1AC3) using Sigma’s Genelute Plasmid Mini-prep kit (sigma) according to the specifications of the manufacturer. Nanodrop utility along with the Spectrophotometer was used to measure the concentration and purity of the isolated plasmid. The wavelength was set at 260nm.

HTML Code, just as an example for now
Plasmid 260/280 260/230 Concentration [ng/μL]
Pqrr4 TOPO Blue tube
1.81
2.24
181
Pqrr4 TOPO Pink tube
1.81
2.02
231.3
Pqrr4 BBK Green tube
1.76
1.66
51.2
Pqrr4 BBK Pink tube 1.85 1.45 60.6

*The two TOPO vectors were from the same colony, thus labelled "Green tube" instead of C1. The two BBK plasmids were also from same colony, so same label applies.


Wiki Code
Table 1. Purity and concentration of Pqrr4 measured with Nanodrop utility and Spectrophotometer

Plasmid 260/280 260/230 Concentration [ng/μL]
Pqrr4 TOPO Blue Tube 1.81 2.24 181
Pqrr4 TOPO Pink Tube 1.81 2.02 231.3
Pqrr4 BBK Green Tube 1.76 1.66 51.2
Pqrr4 BBK Pink Tube 1.85 1.45 60.6


PATRICK

What's in a (DNA Part) Name?

Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.

Continued developing the script for sending names from one part to the next, so that each could learn where the other was. The problem was that each DNA part in a polymer needs to know the key of the next part, in order to direct the RNA Polymerase to the next part when it visits the current one. Yet object keys weren't stable between sessions, so we needed some additional internal name for each part to go by. Each part would know its internal name, and the name of the next part in the sequence. Then it can use that name to obtain the key of the next part via a message and reply.

I first began to use comma separated values (CSV) to encode my messages, which worked so well I never needed to change it. This is thanks to some good built in tools for CSV handling in the linden scripting language.


PRIMA

Back from Dragon's Den

We returned home on the evening of May 26th, weary from the experience and yet somehow craving more of it. Having satisfied both of our original ambitions, we were pleased with the result and thankful that we were more than just another meal for a few Dragons in the vicinity. Armed with a new sense of promotional direction, more contacts who can help support our initiatives, and the same stunning good looks that we had before we left, we are excited to implement some of these ideas to help capitalize on our marketing potential.


VICKI

Plasmid isolation with mini-prep

Purpose:
To isolate the plasmid DNA from the rest of the bacteria for colonies containing LuxPQ on TOPO II Blunt and LuxPQ BBk on psB1AC3.


Reference: The user manual and the Sigma Aldrich site


Kit: MP70 GeneElute Plasmid mini-prep kit. Note that it had been previously used, which may be a problem for contamination.


Materials and methods:
Procedure was carried out in accordance with our Plasmid Isolation protocol. When complete, we stored our isolated plasmids in the freezer for concentration measurement and content analysis over the next 2 days.


Results:
We were left with tubes containing the described plasmids. Quantifiable details on their concentrations and actual contents are outlined on the May 27 and May 28 entries.


DNA concentration measurements with the Nanodrop spectrophotometer

Purpose:
To measure the DNA concentration of LuxPQ plasmids that were isolated in the May 26 miniprep


Materials and Methods:
The DNA measurement area was cleaned with isopropanol and KimWipes to clear off any trace residue from previous uses. The sample wavelength was set to 260 nm. A blank measurement was made with 1 uL of ddHOH. 1 uL of the sample in question was added to the Nanodrop measurement area. The measurement was made; the sample area was wiped down; and a new sample was measured.


Results:

Plasmid 260/280 260/230 Concentration [ng/μL]
LuxPQ TOPO C1 1.83 1.86 338.2
LuxPQ TOPO C2 1.81 1.87 284.2
LuxPQ BBK C1 1.90 2.34 295.5
LuxPQ BBK C2 1.90 2.29 313.2

Retrieved from "http://2009.igem.org/Team:Calgary/26_May_2009"