Team:TMU-Tokyo/Notebook/Experiment/3rd week (8.27 - 9. 2)

From 2012.igem.org

(Difference between revisions)
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<p class="description">
<p class="description">
<b>■3rd week (8.27 - 9. 2)</b><Br></p>
<b>■3rd week (8.27 - 9. 2)</b><Br></p>
 +
<Br>
<p class="description3">
<p class="description3">
-
27th Aug<br />
+
<b>■27th Aug</b><br />
-
Refine of PCR products of frmR and fdh4AB.<br />
+
<b>・Refine of PCR products of frmR and fdh4AB</b><br />
Mixed 1 volume of sample with 2 volumes of Buffer NT.<br />
Mixed 1 volume of sample with 2 volumes of Buffer NT.<br />
Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.<br />
Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.<br />
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Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 45 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<br />
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 45 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<br />
<br />
<br />
-
Densitometry<br />
+
<b>・Densitometry</b><br />
Diluted DNA samples 50 times with a solvent.<br />
Diluted DNA samples 50 times with a solvent.<br />
Turned on the machine; GeneQuant 100.<br />
Turned on the machine; GeneQuant 100.<br />
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Measured the concentration. <br />
Measured the concentration. <br />
<br />
<br />
-
Results<br />
+
<b>・Results</b><br />
Concentration of frmR was 85 ng/ μl.<br />
Concentration of frmR was 85 ng/ μl.<br />
fdh4AB was 43 ng/ μl.<br />
fdh4AB was 43 ng/ μl.<br />
<br />
<br />
-
Gel extraction<br />
+
<b>・Gel extraction</b><br />
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.<br />
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.<br />
Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.<br />
Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.<br />
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Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<br />
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<br />
<br />
<br />
-
Densitometry<br />
+
<b>・Densitometry</b><br />
Concentration of frmR was 53 ng/ μl.<br />
Concentration of frmR was 53 ng/ μl.<br />
fdh4AB was 45 ng/ μl.<br />
fdh4AB was 45 ng/ μl.<br />
<br />
<br />
-
Digestion of frmR and fdh4AB.<br />
+
<b>・Digestion of frmR and fdh4AB</b><br />
1. Mixed following<br />
1. Mixed following<br />
milliQ 5.5 μl<br />
milliQ 5.5 μl<br />
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fdh4AB was 32 ng/ μl.<br />
fdh4AB was 32 ng/ μl.<br />
<br />
<br />
-
28th Aug<br />
+
<Br>
 +
<b>■28th Aug</b><br />
Digestion of backbone plasmid.<br />
Digestion of backbone plasmid.<br />
1. Mixed following<br />
1. Mixed following<br />
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Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<br />
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.<br />
<br />
<br />
-
Densitometry<br />
+
<b>・Densitometry</b><br />
Diluted DNA samples 50 times with a solvent.<br />
Diluted DNA samples 50 times with a solvent.<br />
Turned on the machine; GeneQuant 100.<br />
Turned on the machine; GeneQuant 100.<br />
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Measured the concentration. <br />
Measured the concentration. <br />
<br />
<br />
-
Results<br />
+
<b>・Results</b><br />
Concentration of backbone plasmid was 30 ng/ μl.<br />
Concentration of backbone plasmid was 30 ng/ μl.<br />
<br />
<br />
-
Ligation of frmR.<br />
+
<b>・Ligation of frmR</b><br />
Mixed following<br />
Mixed following<br />
Vector DNA 1 μl<br />
Vector DNA 1 μl<br />
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Total          5 μl
Total          5 μl
<br />
<br />
-
Ligation of fdh4AB.<br />
+
<b>・Ligation of fdh4AB</b><br />
Mixed following<br />
Mixed following<br />
Vector DNA 1 μl<br />
Vector DNA 1 μl<br />
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Incubated at room temperature for 30 minutes.<br />
Incubated at room temperature for 30 minutes.<br />
<br />
<br />
-
Transformation<br />
+
<b>・Transformation</b><br />
Melt competent cells on ice.<br />
Melt competent cells on ice.<br />
Poured DNA solution 10 μl into competent cells calmly.<br />
Poured DNA solution 10 μl into competent cells calmly.<br />
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Plated on an agar medium, and then incubated at 37 °C for overnight.<br />
Plated on an agar medium, and then incubated at 37 °C for overnight.<br />
<br />
<br />
-
Results<br />
+
<b>・Results</b><br />
Colony didn’t appear.<br />
Colony didn’t appear.<br />
<br />
<br />
-
29th Aug<br />
+
<Br>
-
Ligation of frmR.<br />
+
<b>■29th Aug</b><br />
 +
<b>・Ligation of frmR</b><br />
Mixed following<br />
Mixed following<br />
Vector DNA 1 μl<br />
Vector DNA 1 μl<br />
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Total          5 μl
Total          5 μl
<br />
<br />
-
Ligation of fdh4AB.<br />
+
<b>・Ligation of fdh4AB</b><br />
Mixed following<br />
Mixed following<br />
Vector DNA 1 μl<br />
Vector DNA 1 μl<br />
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Incubated at room temperature for 15 minutes.<br />
Incubated at room temperature for 15 minutes.<br />
<br />
<br />
-
Transformation<br />
+
<b>・Transformation</b><br />
Melt competent cells on ice.<br />
Melt competent cells on ice.<br />
Poured DNA solution 10 μl into competent cells calmly.<br />
Poured DNA solution 10 μl into competent cells calmly.<br />
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Plated on an agar medium, and then incubated at 37 °C for overnight.<br />
Plated on an agar medium, and then incubated at 37 °C for overnight.<br />
<br />
<br />
-
Results<br />
+
<b>・Results</b><br />
Colony didn’t appear.<br />
Colony didn’t appear.<br />
<br />
<br />
-
30th Aug<br />
+
<Br>
-
Ligation of frmR.<br />
+
<b>■30th Aug</b><br />
 +
<b>・Ligation</b><Br>
 +
frmR<br />
Mixed following<br />
Mixed following<br />
Vector DNA 1 μl<br />
Vector DNA 1 μl<br />
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Total          5 μl
Total          5 μl
<br />
<br />
-
Ligation of fdh4AB.<br />
+
fdh4AB<br />
Mixed following<br />
Mixed following<br />
Vector DNA 1 μl<br />
Vector DNA 1 μl<br />
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Incubated at room temperature for 10 minutes.<br />
Incubated at room temperature for 10 minutes.<br />
<br />
<br />
-
Transformation<br />
+
<b>・Transformation</b><br />
Melt competent cells on ice.<br />
Melt competent cells on ice.<br />
Poured DNA solution 10 μl into competent cells calmly.<br />
Poured DNA solution 10 μl into competent cells calmly.<br />
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Plated on an agar medium, and then incubated at 37 °C for overnight.<br />
Plated on an agar medium, and then incubated at 37 °C for overnight.<br />
<br />
<br />
-
Results<br />
+
<b>・Results</b><br />
Colony didn’t appear.<br />
Colony didn’t appear.<br />

Revision as of 17:00, 24 September 2012

 




■Experiment
1st week (8.13 - 8.19)
2nd week (8.20 - 8.26)
3rd week (8.27 - 9. 2)
4th week (9. 3 - 9. 9)
5th week (9.10 - 9.16)
6th week (9.17 - 9.23)
7th week (9.24 - 9.30)


■Protocols
Plasmid DNA Purification
Genome DNA Purification
Restruction Enzyme Degestion
DNA Fragment Ligation
Transformation
Electrophoresis
LB Medium


■Assay
Device1 Assay
Device2 Assay
Device3 Assay




Experiment



■3rd week (8.27 - 9. 2)


■27th Aug
・Refine of PCR products of frmR and fdh4AB
Mixed 1 volume of sample with 2 volumes of Buffer NT.
Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Added 700 μl Buffer NT3 to the NucleoSpin Extract II Column. Centrifuged for 1 minute at 11,000 x g. Discarded flow-though and placed the column back into the collection tube.
Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 45 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.

・Densitometry
Diluted DNA samples 50 times with a solvent.
Turned on the machine; GeneQuant 100.
Poured the solvent 100 μl into a cuvette and adjusted 0.
Threw the solvent, poured the DNA sample.
Measured the concentration.

・Results
Concentration of frmR was 85 ng/ μl.
fdh4AB was 43 ng/ μl.

・Gel extraction
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.
Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Added 700 μl Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.

・Densitometry
Concentration of frmR was 53 ng/ μl.
fdh4AB was 45 ng/ μl.

・Digestion of frmR and fdh4AB
1. Mixed following
milliQ 5.5 μl
DNA solution 20 μl
0.5 x K buffer 2.5 μl
SacI 1 μl
BamHI 1 μl Total 50 μl
2. Incubated at 37 °C for 2 hours.

Gel extraction

Densitometry
Concentration of frmR was 45 ng/ μl.
fdh4AB was 32 ng/ μl.


■28th Aug
Digestion of backbone plasmid.
1. Mixed following
milliQ 3.55 μl
DNA solution 18.2 μl
0.5 x K buffer 1.25 μl
SacI 1 μl
BamHI 1 μl Total 25 μl
2. Incubated at 37 °C for 2 hours.

Gel extraction
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.
Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Added 700 μl Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 1 minute at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Centrifuged for 2 minutes at 11,000 x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.

・Densitometry
Diluted DNA samples 50 times with a solvent.
Turned on the machine; GeneQuant 100.
Poured the solvent 100 μl into a cuvette and adjusted 0.
Threw the solvent, poured the DNA sample.
Measured the concentration.

・Results
Concentration of backbone plasmid was 30 ng/ μl.

・Ligation of frmR
Mixed following
Vector DNA 1 μl
Insert DNA 1.6 μl
TE 2.4 μl Total 5 μl
・Ligation of fdh4AB
Mixed following
Vector DNA 1 μl
Insert DNA 2.3 μl
TE 2.7 μl Total 6 μl
Added Ligation Mix 5 μl.
Incubated at room temperature for 30 minutes.

・Transformation
Melt competent cells on ice.
Poured DNA solution 10 μl into competent cells calmly.
Incubated on the ice for 40 minutes.
Plated on an agar medium, and then incubated at 37 °C for overnight.

・Results
Colony didn’t appear.


■29th Aug
・Ligation of frmR
Mixed following
Vector DNA 1 μl
Insert DNA 1.6 μl
TE 2.4 μl Total 5 μl
・Ligation of fdh4AB
Mixed following
Vector DNA 1 μl
Insert DNA 2.3 μl
TE 2.7 μl Total 6 μl
Added Ligation Mix 5 μl.
Incubated at room temperature for 15 minutes.

・Transformation
Melt competent cells on ice.
Poured DNA solution 10 μl into competent cells calmly.
Incubated on the ice for 40 minutes.
Heat shocked at 42 °C for 30 seconds.
Plated on an agar medium, and then incubated at 37 °C for overnight.

・Results
Colony didn’t appear.


■30th Aug
・Ligation
frmR
Mixed following
Vector DNA 1 μl
Insert DNA 1.6 μl
TE 2.4 μl Total 5 μl
fdh4AB
Mixed following
Vector DNA 1 μl
Insert DNA 2.3 μl
TE 1.7 μl Total 5 μl
3. Added Ligation Mix 5 μl.
Incubated at room temperature for 10 minutes.

・Transformation
Melt competent cells on ice.
Poured DNA solution 10 μl into competent cells calmly.
Incubated on the ice for 40 minutes.
Heat shocked at 42 °C for 30 seconds
Incubated on the ice for 2 minutes.
Plated on an agar medium, and then incubated at 37 °C for overnight.

・Results
Colony didn’t appear.