Difference between revisions of "Team:Munich/purify1.html"

 
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<h4>T7 DNA extraction by phenol chloroform precipitation </h4>
+
<h4>T7 DNA Extraction by Phenol Chloroform Precipitation </h4>
 
<em>2018/05/17</em>
 
<em>2018/05/17</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> still too much from E. coli
+
       <td> Still too much DNA from E. coli
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td> DNA concentration: 55,5 ng/µL
 
       <td> DNA concentration: 55,5 ng/µL
 
</td>
 
</td>
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</table>
 
</table>
 
   
 
   
−
<figure class="figure">
+
 
−
  <img src="SOURCE" class="figure-img img-fluid rounded" alt="error">
+
−
    <figcaption class="figure-caption">0,3% Agarose gel, 2 log ladder </figcaption>
+
−
    </figure> HIER FOTO
+
  
 
   
 
   
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> warming of columns on 40 °C for a few minutes before elution </td>
+
       <td> Warming of columns on 40 °C for a few minutes before elution </td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> no results, not possible with genome larger than 25kb
+
       <td> No results, not possible with genome larger than 25kb
 
</td>
 
</td>
 
     </tr>
 
     </tr>
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−
<h4>second try: Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit </h4>
+
<h4>Second Try: Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit </h4>
 
<em>2018/05/20</em>
 
<em>2018/05/20</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> warming of columns on 50 °C for a few minutes before elution </td>
+
       <td> Warming of columns on 50 °C for a few minutes before elution </td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> no results, did not work out for large fragments
+
       <td> No visible bands on 0,3 Agarose gel, did not work out for large fragments. <br>
 +
End of experiments with this method
 
</td>
 
</td>
 
     </tr>
 
     </tr>
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−
<h4>T7 DNA clean up T7 DNA </h4>
+
<h4>T7 DNA Clean-Up T7 DNA </h4>
 
<em>2018/05/22</em>
 
<em>2018/05/22</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
        
−
      <td> no results à  testing PEG cutoff tomorrow
+
−
</td>
+
 
     </tr>
 
     </tr>
 
</table>
 
</table>
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−
<h4> Generating MS2 RNA genome </h4>
+
<h4> Generating MS2 RNA Genome </h4>
 
<em>2018/05/23</em>
 
<em>2018/05/23</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td> RNA concentration: 34900 ng/µl
 
       <td> RNA concentration: 34900 ng/µl
 
</td>
 
</td>
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−
<h4> Generating MS2 RNA genome </h4>
+
<h4> Generating MS2 RNA Genome </h4>
 
<em>2018/05/23</em>
 
<em>2018/05/23</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td> RNA concentration: 34900 ng/µl
 
       <td> RNA concentration: 34900 ng/µl
 
</td>
 
</td>
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</table>
 
</table>
  
−
<h4> Optimization of DNA precipitation out of T7 Phage </h4>
+
<h4> Optimization of DNA Precipitation out of T7 Phage </h4>
 
<em>2018/05/23 - 2018/05/25</em>
 
<em>2018/05/23 - 2018/05/25</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td>  150 µl PEG: 302 ng/µl, 200 µl PEG: 164 ng/µl, 250 µl PEG: 278 ng/µl, 300 µl PEG: 113 ng/µl, 350 µl PEG: 279 ng/µl, 400 µl PEG: 216 ng/µl, 450 µl PEG: 530 ng/µl,  500 µl PEG: 422 ng/µl
 
       <td>  150 µl PEG: 302 ng/µl, 200 µl PEG: 164 ng/µl, 250 µl PEG: 278 ng/µl, 300 µl PEG: 113 ng/µl, 350 µl PEG: 279 ng/µl, 400 µl PEG: 216 ng/µl, 450 µl PEG: 530 ng/µl,  500 µl PEG: 422 ng/µl
 
</td>
 
</td>
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−
<h4>T7 DNA Isolation with different infection times </h4>
+
<h4>T7 DNA Isolation with Different Infection Times </h4>
 
<em>2018/05/29</em>
 
<em>2018/05/29</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> tested transfection times: 12 min (before supernatant), 22 min (after supernatant), 32 min (cell lysis)
+
       <td> Tested transfection times: 12 min (before supernatant), 22 min (after supernatant), 32 min (cell lysis)
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td> 12 min:  209 ng/µL, 22 min:  628 ng/µL, 32 min:  334 ng/µL
 
       <td> 12 min:  209 ng/µL, 22 min:  628 ng/µL, 32 min:  334 ng/µL
 
</td>
 
</td>
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−
<h4> T7 DNA Isolation 10 min and 32 min after super transfection + RNAse </h4>
+
<h4> T7 DNA Isolation 10 min and 32 min after Super Transfection + RNAse </h4>
 
<em>2018/06/05 -2018/06/06</em>
 
<em>2018/06/05 -2018/06/06</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> better results than DNA without RNAse treatment, still too much E. coli DNA
+
       <td> Better results than DNA without RNAse treatment, still too much E. coli DNA
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td> DNA concentration of 10 min: 633 ng/µL
 
       <td> DNA concentration of 10 min: 633 ng/µL
 
DNA concentration of 32 min:  200 ng/µL
 
DNA concentration of 32 min:  200 ng/µL
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</table>
 
</table>
  
−
<figure class="figure">
+
 
−
  <img src="SOURCE" class="figure-img img-fluid rounded" alt="error">
+
−
    <figcaption class="figure-caption">0,3% Agarose gel, 2 log ladder, after 10 min infection</figcaption>
+
−
    </figure> HIER FOTO
+
−
+
−
<figure class="figure">
+
−
  <img src="SOURCE" class="figure-img img-fluid rounded" alt="error">
+
−
    <figcaption class="figure-caption">0,3% Agarose gel, 2 log ladder, after 10 min infection (upper picture) and 32 min infection</figcaption>
+
−
    </figure> HIER FOTO
+
 
   
 
   
−
<h4>T7 DNA extraction by phenol chloroform precipitation </h4>
+
<h4>T7 DNA Extraction by Phenol Chloroform Precipitation </h4>
 
<em>2018/06/16-2018/06/18</em>
 
<em>2018/06/16-2018/06/18</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> -
+
       <td> No notes
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> good DNA: 10,37 nM
+
       <td> Good DNA: 10,37 nM
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
−
<h4> DNA purification of T7 DNA with Qiagen Genomic Tips 100/G</h4>
+
<h4> DNA Purification of T7 DNA with Qiagen Genomic Tips 100/G</h4>
 
<em>2018/06/22</em>
 
<em>2018/06/22</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> preparation of buffers as descripted on their homepage
+
       <td> Preparation of buffers as descripted on their homepage
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> did not work out
+
       <td> Did not work out
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
 
   
 
   
−
<h4> DNA purification of T7 DNA with Qiagen Genomic Tips 100/G</h4>
+
<h4> DNA Purification of T7 DNA with Qiagen Genomic Tips 100/G</h4>
 
<em>2018/06/25</em>
 
<em>2018/06/25</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td> increased DNA concentration 50 ug
+
       <td> Increased DNA concentration 50 ug
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> did not work out
+
       <td> Did not work out. <br>
 +
Method not suitable for phage DNA.
 
</td>
 
</td>
 
     </tr>
 
     </tr>

Latest revision as of 02:22, 18 October 2018

T7 DNA Extraction by Phenol Chloroform Precipitation

2018/05/17
Participants: Keno Eilers
Protocol: Phage T7 DNA Purification, Phenol Chloroform precipitation
Notes: Still too much DNA from E. coli
Results: DNA concentration: 55,5 ng/µL

Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit

2018/05/19
Participants: Nils O'Brien
Protocol: NEB PCR Cleanup Kit, Agarose Gel electrophoresis
Notes: Warming of columns on 40 °C for a few minutes before elution
Results: No results, not possible with genome larger than 25kb

Second Try: Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit

2018/05/20
Participants: Nils O'Brien
Protocol: NEB PCR Cleanup Kit, Agarose Gel electrophoresis
Notes: Warming of columns on 50 °C for a few minutes before elution
Results: No visible bands on 0,3 Agarose gel, did not work out for large fragments.
End of experiments with this method

T7 DNA Clean-Up T7 DNA

2018/05/22
Participants: Keno Eilers
Protocol: Phenol Chloroform precipitation

Generating MS2 RNA Genome

2018/05/23
Participants: Keno Eilers
Protocol: Phenol Chloroform precipitation, cDNA Synthesis
Notes: 1 µl DNAse I (NEB, 2.000 U/mL) added, incubate for 10 minutes at 37 °C
Results: RNA concentration: 34900 ng/µl

Generating MS2 RNA Genome

2018/05/23
Participants: Keno Eilers
Protocol: Phenol Chloroform precipitation, cDNA Synthesis
Notes: 1 µl DNAse I (NEB, 2.000 U/mL) added, incubate for 10 minutes at 37 °C
Results: RNA concentration: 34900 ng/µl

Optimization of DNA Precipitation out of T7 Phage

2018/05/23 - 2018/05/25
Participants: Keno Eilers, Sophie Kurzbach, Nils O'Brien
Protocol: Phage T7 DNA Purification, Agarose Gelelectrophoresis
Notes: Step 11 of "T7 DNA purification" protocol: Optimization of PEG (63%) precipitation Incubation for 42 minutes, then proceeded with centrifugation
Results: 150 µl PEG: 302 ng/µl, 200 µl PEG: 164 ng/µl, 250 µl PEG: 278 ng/µl, 300 µl PEG: 113 ng/µl, 350 µl PEG: 279 ng/µl, 400 µl PEG: 216 ng/µl, 450 µl PEG: 530 ng/µl, 500 µl PEG: 422 ng/µl

T7 DNA Isolation with Different Infection Times

2018/05/29
Participants: Sophie Kurzbach
Protocol: Phage T7 DNA Purification, Phenol Chloroform precipitation
Notes: Tested transfection times: 12 min (before supernatant), 22 min (after supernatant), 32 min (cell lysis)
Results: 12 min: 209 ng/µL, 22 min: 628 ng/µL, 32 min: 334 ng/µL

T7 DNA Isolation 10 min and 32 min after Super Transfection + RNAse

2018/06/05 -2018/06/06
Participants: Sophie Kurzbach
Protocol: Phage T7 DNA Purification, Phenol Chloroform precipitation, PEG percipitant
Notes: Better results than DNA without RNAse treatment, still too much E. coli DNA
Results: DNA concentration of 10 min: 633 ng/µL DNA concentration of 32 min: 200 ng/µL

T7 DNA Extraction by Phenol Chloroform Precipitation

2018/06/16-2018/06/18
Participants: Keno Eilers
Protocol: Phage T7 DNA Purification, Phenol Chloroform precipitation
Notes: No notes
Results: Good DNA: 10,37 nM

DNA Purification of T7 DNA with Qiagen Genomic Tips 100/G

2018/06/22
Participants: Brigit Tunaj
Protocol: Qiagen Genomic Tip Kit
Notes: Preparation of buffers as descripted on their homepage
Results: Did not work out

DNA Purification of T7 DNA with Qiagen Genomic Tips 100/G

2018/06/25
Participants: Brigit Tunaj
Protocol: Qiagen Genomic Tip Kit
Notes: Increased DNA concentration 50 ug
Results: Did not work out.
Method not suitable for phage DNA.