Team:Evry/Notebook/Interlabstudy/Week7
From 2014.igem.org
(Difference between revisions)
Line 19: | Line 19: | ||
<div class="thumbinner" style="width:502px;"> | <div class="thumbinner" style="width:502px;"> | ||
<a href="URL IMAGE" class="image"> | <a href="URL IMAGE" class="image"> | ||
- | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/a8/PCR12082014.jpg" width=" | + | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/a8/PCR12082014.jpg" width="400px;" class="thumbimage"/> |
</a> | </a> | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
Line 38: | Line 38: | ||
<div class="thumb tnone"> | <div class="thumb tnone"> | ||
<div class="thumbinner" style="width:502px;"> | <div class="thumbinner" style="width:502px;"> | ||
- | <a href=" | + | <a href="https://static.igem.org/mediawiki/2014/5/5f/Thermocycling_cond_Q5.jpg" class="image"> |
- | <img alt="IMAGE" src=" | + | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/5/5f/Thermocycling_cond_Q5.jpg" width="200px;" class="thumbimage"/> |
</a> | </a> | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<div class="magnify"> | <div class="magnify"> | ||
- | <a href=" | + | <a href="https://static.igem.org/mediawiki/2014/5/5f/Thermocycling_cond_Q5.jpg" class="internal" title="Enlarge"> |
<img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/> | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/> | ||
</a> | </a> | ||
Line 63: | Line 63: | ||
<div class="thumbinner" style="width:502px;"> | <div class="thumbinner" style="width:502px;"> | ||
<a href="URL IMAGE" class="image"> | <a href="URL IMAGE" class="image"> | ||
- | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/b/bf/Gel12082014.jpg" width=" | + | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/b/bf/Gel12082014.jpg" width="400px;" class="thumbimage"/> |
</a> | </a> | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
Line 95: | Line 95: | ||
<div class="thumb tnone"> | <div class="thumb tnone"> | ||
<div class="thumbinner" style="width:502px;"> | <div class="thumbinner" style="width:502px;"> | ||
- | <a href=" | + | <a href="/wiki/images/0/0a/Gel13082014.jpg" class="image"> |
- | <img alt="IMAGE" src="/wiki/images/0/0a/Gel13082014.jpg" width=" | + | <img alt="IMAGE" src="/wiki/images/0/0a/Gel13082014.jpg" width="400px;" class="thumbimage"/> |
</a> | </a> | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
Line 111: | Line 111: | ||
<br/> | <br/> | ||
- | Profiles were same as before PCR clean up. | + | Profiles were same as before PCR clean up. To sequence the amplified parts, we decided to a purification of fragments from the gel as presented below. |
<br/> | <br/> | ||
<br/>Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. | <br/>Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. | ||
<br/>Microwave 30s by 30s until agarose total dissolution | <br/>Microwave 30s by 30s until agarose total dissolution | ||
Gel was cooling down until to be lukewarm, one BET drop was added. | Gel was cooling down until to be lukewarm, one BET drop was added. | ||
- | Gel was loaded with 30 µl of BBa_E0240 purified PCR product and BBa_I20260 purified PCR product added with 6 µl of loading dye. | + | Gel was loaded with 30 µl of BBa_E0240 purified PCR product and BBa_I20260 purified PCR product added with 6 µl of loading dye, at 10µl of the mix per well. |
Gel running 45 minutes at 100 mV in TAE 1X buffer. | Gel running 45 minutes at 100 mV in TAE 1X buffer. | ||
Line 123: | Line 123: | ||
<div class="thumbinner" style="width:502px;"> | <div class="thumbinner" style="width:502px;"> | ||
<a href="URL IMAGE" class="image"> | <a href="URL IMAGE" class="image"> | ||
- | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" width=" | + | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" width="300px;" class="thumbimage"/> |
</a> | </a> | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
Line 141: | Line 141: | ||
<h2> 08.14.2014 </h2> | <h2> 08.14.2014 </h2> | ||
+ | To amplify BBa_I20260 and BBa_0240, a PCR was perform with the mix described Table 1. The program IGEM Q5 PCR was applied, see Table 2. | ||
+ | <br/> PCR products were cleaned with the GeneJET purification kit (Thermo Scientific). | ||
+ | <br/> DNA quantification with the NanoDrop 2000 (Thermo Scientific). | ||
+ | |||
+ | |||
+ | <br/>Preparation of a 1% agarose gel: 0.52 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. | ||
+ | <br/>Microwave 30s by 30s until agarose total dissolution | ||
+ | Gel was cooling down until to be lukewarm, one BET drop was added. | ||
+ | Gel was loaded with 20 µl of BBa_E0240 PCR product and BBa_I20260 purified PCR product added with 4 µl of loading dye, at 10µl of the mix per well. | ||
+ | Gel running 45 minutes at 100 mV in TAE 1X buffer. | ||
+ | |||
+ | |||
+ | <br/>3 pieces of gel were sampled in four tubes to perform a DNA purification from gel. | ||
+ | |||
+ | <h2> 08.15.2014 </h2> | ||
+ | |||
+ | |||
+ | <h2> 08.16.2014 </h2> | ||
+ | |||
+ | <h2> 08.17.2014 </h2> | ||
</p> | </p> |
Revision as of 08:13, 18 August 2014
Week 7
Interlab Study
08.12.2014
The 4 needed parts are: BBa_J23101, BBa_J23115, BBa_E0240 and BBa_I20260. Corresponding wells were located on 2014 Distribution kit plates and resuspended with 10 µL steril water. That permits to obtain a DNA concentration around 0.2 ng/µl (according to the registry. Solutions were transferred into 1 ml eppendorf tubes and stored at -20°C.
To amplify fragments, a PCR was performed on the 4 constructions, with the mix described on table 1.
Distribution of 49 µl of mix per PCR tube. Application of program IGEM Q5 PCR.
Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution
Gel was cooling down until to be lukewarm, one BET drop was added.
Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders.
Gel running 45 minutes at 100 mV in TAE 1X buffer.
We expected to obtain one band per PCR sample corresponding to the interesting amplified fragment.
For Lane 2, 3 and 6 it was ok. We had the expected profile. By contrast 2 bands were visible, one at the expected size (around 1200 bp) and another around 600 bp. We decided to perform a purification on gel of each bands.
08.13.2014
PCR products were cleaned with the GeneJET purification kit (Thermo Scientific).
DNA quantification with the NanoDrop 2000 (Thermo Scientific).
Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution
Gel was cooling down until to be lukewarm, one BET drop was added.
Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders.
Gel running 45 minutes at 100 mV in TAE 1X buffer.
Profiles were same as before PCR clean up. To sequence the amplified parts, we decided to a purification of fragments from the gel as presented below.
Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution
Gel was cooling down until to be lukewarm, one BET drop was added.
Gel was loaded with 30 µl of BBa_E0240 purified PCR product and BBa_I20260 purified PCR product added with 6 µl of loading dye, at 10µl of the mix per well.
Gel running 45 minutes at 100 mV in TAE 1X buffer.
Four pieces of gel were sampled in four tubes to perform a DNA purification from gel.
08.14.2014
To amplify BBa_I20260 and BBa_0240, a PCR was perform with the mix described Table 1. The program IGEM Q5 PCR was applied, see Table 2.
PCR products were cleaned with the GeneJET purification kit (Thermo Scientific).
DNA quantification with the NanoDrop 2000 (Thermo Scientific).
Preparation of a 1% agarose gel: 0.52 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution
Gel was cooling down until to be lukewarm, one BET drop was added.
Gel was loaded with 20 µl of BBa_E0240 PCR product and BBa_I20260 purified PCR product added with 4 µl of loading dye, at 10µl of the mix per well.
Gel running 45 minutes at 100 mV in TAE 1X buffer.
3 pieces of gel were sampled in four tubes to perform a DNA purification from gel.
08.15.2014
08.16.2014
08.17.2014
Interlab Study
08.12.2014
The 4 needed parts are: BBa_J23101, BBa_J23115, BBa_E0240 and BBa_I20260. Corresponding wells were located on 2014 Distribution kit plates and resuspended with 10 µL steril water. That permits to obtain a DNA concentration around 0.2 ng/µl (according to the registry. Solutions were transferred into 1 ml eppendorf tubes and stored at -20°C.
To amplify fragments, a PCR was performed on the 4 constructions, with the mix described on table 1.
Distribution of 49 µl of mix per PCR tube. Application of program IGEM Q5 PCR. Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders. Gel running 45 minutes at 100 mV in TAE 1X buffer.
We expected to obtain one band per PCR sample corresponding to the interesting amplified fragment. For Lane 2, 3 and 6 it was ok. We had the expected profile. By contrast 2 bands were visible, one at the expected size (around 1200 bp) and another around 600 bp. We decided to perform a purification on gel of each bands.
08.13.2014
PCR products were cleaned with the GeneJET purification kit (Thermo Scientific).DNA quantification with the NanoDrop 2000 (Thermo Scientific).
Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders. Gel running 45 minutes at 100 mV in TAE 1X buffer.
Profiles were same as before PCR clean up. To sequence the amplified parts, we decided to a purification of fragments from the gel as presented below.
Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 30 µl of BBa_E0240 purified PCR product and BBa_I20260 purified PCR product added with 6 µl of loading dye, at 10µl of the mix per well. Gel running 45 minutes at 100 mV in TAE 1X buffer.
Four pieces of gel were sampled in four tubes to perform a DNA purification from gel.
08.14.2014
To amplify BBa_I20260 and BBa_0240, a PCR was perform with the mix described Table 1. The program IGEM Q5 PCR was applied, see Table 2.PCR products were cleaned with the GeneJET purification kit (Thermo Scientific).
DNA quantification with the NanoDrop 2000 (Thermo Scientific).
Preparation of a 1% agarose gel: 0.52 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 20 µl of BBa_E0240 PCR product and BBa_I20260 purified PCR product added with 4 µl of loading dye, at 10µl of the mix per well. Gel running 45 minutes at 100 mV in TAE 1X buffer.
3 pieces of gel were sampled in four tubes to perform a DNA purification from gel.