Difference between revisions of "Team:Rheda Bielefeld/Notebook"

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<div class="header">
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<img src="https://static.igem.org/mediawiki/2018/f/f1/T--Rheda_Bielefeld--dq.jpg" style="width:50%;height:auto;"></img>
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</div>
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<div class="row">
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<h2> Notebook </h2>
 +
<article> Wet-Lab Protocol <br><br>
 +
 +
22.05.2018 <br>
 +
Opening pollen with Trypsin <br>
 +
-Resuspending 20µg Trypsin in 200 µl of water <br>
 +
-Splitting solution in 4 eppis with 50 µl each => approxamittly 5 µg of Trypsin in each eppi <br>
  
−
<div class="column full_size">
+
-5000 U/mg=Proportionalitycalculation <br>
  
−
<h1>Notebook</h1>
+
-50mg of pollenmaterial <br>
−
<p> Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.</p>
+
  
−
</div>
+
-3 active Trypsin-eppis (incubating at 30 °C for 15 minutes) <br>
−
<div class="clear"></div>
+
  
 +
-Approxamittly 15 µg of Trypsin available <br>
  
 +
Calculating units: <br>
  
−
<div class="column two_thirds_size">
+
-1 mg= 5000 U <br>
−
<h3>What should this page have?</h3>
+
−
<ul>
+
−
<li>Chronological notes of what your team is doing.</li>
+
−
<li> Brief descriptions of daily important events.</li>
+
−
<li>Pictures of your progress. </li>
+
−
<li>Mention who participated in what task.</li>
+
−
</ul>
+
  
−
</div>
+
-1 solution: 15 µg= 0.015 mg= 75 U=> 1:3.3 <br>
  
−
<div class="column third_size">
+
-2 solutions: 7.5 µg=0.0075 mg= 37 U=> 1:6.6 <br>
−
<div class="highlight decoration_A_full">
+
−
<h3>Inspiration</h3>
+
−
<p>You can see what others teams have done to organize their notes:</p>
+
  
−
<ul>  
+
-3 solutions: 5 µg=0.005 mg= 25 U => 1:10<br>
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+
-Proportionality=weight Trypsin : weight pollen <br>
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+
 
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+
-Add 50 µg of pollen to 10µl  Trypsin and 120µl Ammoniumhydrocarbonate <br>
−
</ul>
+
 
 +
-Incubating at 37°C over night <br>
 +
 
 +
=> analyzed with light microscopy on 23.05.: did not work <br><br>
 +
 
 +
 
 +
-Microscopy Birchpollen (100x) <br>
 +
 
 +
-Microscopy Birchpollen (400x) <br>
 +
 
 +
-Microscopy Willowpollen (100x) <br>
 +
 
 +
-Microscopy Willowpollen (400x) <br>
 +
 
 +
-Microscopy Sprucepollen (100x) <br>
 +
 
 +
-Microscopy Sprucepollen (400x) <br><br>
 +
 
 +
 
 +
 +
Extraction of pollen <br>
 +
 
 +
-Putting male infructescence into a electrostaticly loaded plastic tube <br>
 +
 
 +
-Vortexing tube => pollen stick to walls of the tube <br>
 +
 
 +
-Extracting pollen <br>
 +
 
 +
-Analyzing with light microscopy if only one type of pollen is in each tube <br>
 +
<br>
 +
 
 +
 +
 +
23.05.2018<br>
 +
<br>
 +
 +
Electromicroscopy <br>
 +
<br>
 +
 
 +
-Prepare samples <br>
 +
 
 +
-> 1: birchpollen <br>
 +
 
 +
-> 2: Spruce-trypsin supernatant <br>
 +
 
 +
-> 3: Spruce-trypsin sediment<br>
 +
 +
-> 4: SPruce and spruce ribolysed <br><br>
 +
 
 +
 
 +
-Dried in etikator<br>
 +
 +
-Evaporated with gold  <br>
 +
 
 +
-DNA-extraction pollen <br>
 +
 
 +
-50 mg in rybolyser tubes <br>
 +
 
 +
-Constant shaking in rybolyser (6500*2*30*30) <br>
 +
 
 +
-> centrifugate for 10 min (1100 rpm)<br><br>
 +
 
 +
 +
Results: <br>
 +
 
 +
-Two layers:<br>
 +
 +
-> over the upper one: white shroud <br>
 +
 
 +
->  light-brown layer of pollen: abstraction with chemical droppper<br>
 +
 +
-> 400µl lysebuffer, centrifugate 1 min (11000 rpm)  <br>
 +
 
 +
-> DNA-extraction: machery nagel plant NUcleo spin III Kit <br><br>
 +
 
 +
 
 +
 +
Nanodrop
 +
Samples from leafes
 +
 
 +
<table style= "width 100%">
 +
<tr>
 +
<th> B </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 22,7 <br> 1,68 <br> 1,23 </th>
 +
<th> 97,2 <br> 1,71 <br> 0,83 </th>
 +
</tr>
 +
<tr>
 +
<th> PA </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
 
 +
<th> 3,1<br> 0,97 <br> 1,05 </th>
 +
 
 +
<th> 8,9 <br> 1,83 <br> 0,83 </th>
 +
</tr>
 +
<tr>
 +
<th> PB </th>
 +
<th> ng/µl<br> 260/280 <br> 260/230 </th>
 +
<th> 30,2 <br> 2,34 <br> 0,73 </th>
 +
<th> 35,1 <br> 1,84 <br> 0,89 </th>
 +
</tr>
 +
</table>
 +
Opening pollen with liquid nitrogen <br>
 +
-Nitrogen and mortar and pestle <br>
 +
-Check: light microscope <br>
 +
Opening pollen with ribolyser <br><br>
 +
-10,20 & 50 mg pollen and 4oo µl water each in ribolyser tube<br>
 +
-Ribolyser (6500-3*45-30) <br>
 +
->Just 10 mg worked (light microscope) <br><br>
 +
 +
24.05.2018 <br><br>
 +
Pollensamples evaporate with gold (10-20 nm)<br>
 +
<table style="width 100%">
 +
<tr>
 +
<th> nitrogen </th>
 +
<th> ng>/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 66,6 <br> 1,88 <br> 1,69 </th>
 +
<th> 75,6<br> 1,71 <br> 1,33 </th>
 +
</tr>
 +
<tr>
 +
<th> trypsin </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 418,2 <br> 1,72<br> 1,00 </th>
 +
<th> 399,2 <br> 1,59 <br> 0,86 </th>
 +
</tr>
 +
</table><br>
 +
 +
 
 +
DNA-extraction of pollen<br>
 +
-1: opened with trypsin <br>
 +
-2: opened with liquid nitrogen <br>
 +
-> 1: 400 µl PL1 and 1 pollen and 10µl RNA-se ( centrifugate 1min 11000 rpm) <br>
 +
-> 2: 800 µl PL1 and 2 pollen and 10 µl RNA-se ( centrifugate 2min 11000 rpm) <br>
 +
-> protocol DNA extraction <br><br>
 +
DNA-extraction from birchleafes <br>
 +
-Protocol <br>
 +
DNA-extraction from spruce needles <br>
 +
-Liquid nitrogen <br><br>
 +
 +
17.07.2018 <br><br>
 +
Extracted DNA: Nanodrop und PCR <br>
 +
-> 5x Phusion HF buffer: 36 µl <br>
 +
-> 10 mn dNTPs: 3.6 µl <br>
 +
-> Fw primer: 9µl <br>
 +
-> rw primer: 9µl <br>
 +
-> template DNA: 9µl <br>
 +
-> Phusion DNA polymerase: 1.8 µl <br>
 +
-> H2o: 111.6 µl <br>
 +
5 primer mixtures: - SP1 <br>
 +
                                        - sp2 V
 +
                                        - ec1    --> each with DNA sample and positive and negative control <br>
 +
                                        -ec2 <br>
 +
                                        -bet  <br><br>
 +
Results Nanodrop <br>
 +
<table style ="width 100%">  
 +
<tr>
 +
<th> birch pollen+ nitrogen </th>
 +
<th> 8ng/µl </th>
 +
 
 +
</tr>
 +
 
 +
<tr>
 +
 
 +
<th> spruce pollen+ nitrogen </th>
 +
 
 +
<th> 10,5 ng/µl </th>
 +
 
 +
</tr>
 +
 
 +
<tr>
 +
 
 +
<th> birch leaves+ mortar </th>
 +
<th> 11,5 ng/µl </th>
 +
</tr>
 +
<tr>
 +
<th> birch leaves+mortar </th>
 +
<th> 5,5 ng/µl </th>
 +
</tr>
 +
<tr>
 +
<th> birch leaves+ mortar </th>
 +
<th> 17,5 ng/µl </th>
 +
</tr>
 +
</table>
 +
 
 +
B. sub
 +
-600µl 5c buffer+ cells from plate <br>
 +
-600 µl in ribolyser tubes <br>
 +
-Ribolyse <br>
 +
-Centrifugate: 5 min, 8000 rpm <br>
 +
-Take supernatant 
 +
+1ml NaCl <br>
 +
-Filtrate <br><br>
 +
Nanodrop B. Sub.
 +
 
 +
<table style ="width 100%">
 +
<tr>
 +
<th> Leon </th>  
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 292,2 <br> 1,97<br> 1,12 </th>
 +
<th> 185,7 <br> 1,59 <br> 0,84 </th>
 +
</tr>
 +
<tr>
 +
<th> Viviane </th>
 +
<th> ng/µl <br> 260/280<br> 260/230 </th>
 +
<th> 324,7 <br> 1,96 <br> 1,16 </th>
 +
<th> 329,8 <br> 1,96 <br> 1,16 </th>
 +
</tr>
 +
<tr>
 +
<th> Jil </th>
 +
<th> ng/µl<br> 260/280 <br> 260/230 </th>
 +
<th> 192,4 <br> 1,98 <br> 1,19 </th>
 +
<th> 263,7 <br> 2,01 <br> 1,19 </th>
 +
</tr>
 +
</table>
 +
 
 +
Nanodrop plasmid <br>
 +
<table style= "width 100%">
 +
<tr>
 +
<th> Fynn </th>
 +
<th> ng/µl <br> 260/280<br> 260/230 </th>
 +
<th> 37,7 <br> 1,96<br> 7,29 </th>
 +
<th> 39,1 <br> 1,94 <br> 5,95 </th>
 +
</tr>
 +
<tr>
 +
<th> Elisa</th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 84,0 <br> 1,93<br> 2,24 </th>
 +
<th> 83,3 <br> 1,97 <br> 1,92 </th>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
Plasmids DNA-isolation: innuprep plasmid mini kit 2.0 <br>
 +
                                          -> protocol 1: step 9: 30µl h2o instead of 50-100µ elution buffer <br>
 +
-> one more time 18.07. <br><br>
 +
 +
18.07.2018 <br><br>
 +
Nanodrop psb1c3 <br>
 +
 
 +
<table style=" width 100%">
 +
<tr>
 +
<th> Jil </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 193,6 <br> 1,89 <br> 2,84 </th>
 +
<th> 192,1<br> 1,89 <br> 1,78 </th>
 +
</tr>
 +
<tr>
 +
<th> Elisa </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 33,4 <br> 2,00 <br> 43,42 </th>
 +
<th> 40,0 <br> 2,07 <br> -14,57 </th>
 +
</tr>
 +
</table> <br>
 +
 
 +
PCR B.Sub. (bsub_pelB) 1st try <br><br>
 +
-PCR Phusion 3 step protocol (without DMSO) <br>
 +
-> fragment size: 1038 bp <br>
 +
-> Tm °C: 62 <br>
 +
-> extension: 20s/kb <br>
 +
-Breeding from pz9-plasmids <br>
 +
-Isolated pz9 plasmids+ <br>
 +
-> phusion HF buffer: 20µl <br>
 +
-> dNTPs: 5µl <br>
 +
-> fw primer:5µl <br>
 +
-> rv primer: 5µl <br>
 +
-> template DNA: 5µl <br>
 +
-> Phusion polymerase: 1µl<br>
 +
-> h20: 62µl <br>
 +
-> fragment size: ca. 5200 bp <br>
 +
-> Tm°C: 59 <br>
 +
-> extension: 2:40 <br><br>
 +
Nanodrop Xan. <br>
 +
<table style = "width 100% ">
 +
<tr>
 +
<th> Ben </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230 </th>
 +
<th> 109,6 <br> 1,99 <br> 1,21 </th>
 +
<th> 122,1 <br> 1,93 <br> 1,23 </th>
 +
</tr>
 +
<tr>
 +
<th> Simon </th>
 +
<th> ng/µl <br> 260/280 <br> 260/230</th>
 +
<th> 109,6 <br> 1,96<br> 1,1 </th>
 +
<th> 111,4 <br> 1,98 <br> 1,15 </th>
 +
</tr>
 +
</table><br> <br>
 +
 +
 +
PCR XAN <br>
 +
-1. primer Xan 1+2 <br>
 +
-2. primer Xan 3+4 <br>
 +
-As breeding pz9  <br>
 +
-> fragment size: 745 bp<br> 
 +
-> Tm°C: 62 <br>
 +
-> 3µl DMSO <br>
 +
-> extension: 30s <br> <br>
 +
PCR B.Sub. 3rd try <br>
 +
-Taq-polymerase  <br>
 +
-> long size: 1038 bp<br> 
 +
-> Tm°C: 59-70 <br>
 +
-> DMS0: 3% <br> <br>
 +
2nd isolation of chromosomal DNA from b.sub. for transformation (standard transformation protocol) <br>
 +
-BBa_JO4450 Trafo <br>
 +
-Promega standard transformation protocol <br>
 +
-Kit 7, 23 O (iGEM) <br>
 +
-50 µl plated <br>
 +
-Centrifugate: 2 min, 5000rpm <br>
 +
-Pellet resuspended, supernatant plated <br> <br>
 +
 +
20.07.2018 <br> <br>
 +
Nanodrop bacillus subtilis <br>
 +
<table style="width 100%">
 +
<tr>
 +
<th> B.Sub. 1</th>
 +
<th> ng/µl <br>  260/280 <br> 260/230 </th>
 +
<th> 93,7 <br>  1,71 <br>  0,68 </th>
 +
<th> 90,8 <br>  1,81 <br>  0,66 </th>
 +
</tr>
 +
<tr>
 +
<th> B.Sub. 2 </th>
 +
<th> ng/µl <br>  260/280 <br> 260/230 </th>
 +
<th> 178,2 <br>  1,74 <br>  0,64 </th>
 +
<th> 176,6 <br>  1,72 <br>  0,66 </th>
 +
</tr>
 +
</table>
 +
<br> 
 +
Nanodrop xanthomonas
 +
 
 +
<table style=" width 100%">
 +
 
 +
<tr>
 +
<th> Xan.th>
 +
<th> ng/µl <br>  260/280<br>  260/230 </th>
 +
<th> 35,2 <br>  1,92 <br>  1,76 </th>
 +
<th> 33,1 <br>  1,83 <br>  1,51 </th>
 +
</tr>
 +
<tr>
 +
<th> Xan.2 </th>
 +
<th> ng/µl<br>  260/280 <br>  260/230 </th>
 +
<th> 40,4 <br>  1,84 <br>  1,81 </th>
 +
<th> 40,2 <br>  1,86 <br>  1,80 </th>
 +
</tr>
 +
</table> <br> <br>
 +
 
 +
 
 +
 
 +
 +
Gelelectrophoresis psb1c3 <br>
 +
-1: 1-4 : E, 5-7: J (kept Dna) <br>
 +
-2: 1-3: E, 4-7: J, 8: size standard (new isolated DNA)  <br> <br>
 +
 +
PCR Fynn B.sub. 2nd  try<br> 
 +
->60,8-70,2 °C <br> <br>
 +
 +
 +
24.07.2018 <br> <br>
 +
PCR pz9 <br>
 +
-MM: 36µl <br>
 +
-H2O: 117µl <br>
 +
-Primer: 9µl <br>
 +
-Template DNA: 9µl <br>
 +
-> gradient PCR: 62,1-68,9 °C <br> <br>
 +
 +
PCR B.Sub. 4th try <br>
 +
-Taq PCR, new DNA <br>
 +
-> approaches : 2x8 <br>
 +
    -Frag size: 1038 bp <br>
 +
    -Tm°C: 60-70 <br>
 +
    -DMSO: 3% <br> <br>
 +
 +
PCR Xan. Ben&Simon <br>
 +
-> Phusion: protocol <br>
 +
-Frag size: 700, 1200<br> 
 +
-Tm°C: 61,5 <br> <br>
 +
 +
Plating trafos<br> 
 +
-50µl plated <br>
 +
-Sample centrifugated 3min, 4000 rpm <br>
 +
-Supernatant removed <br>
 +
-Resuspend pellet <br>
 +
-Plate  <br> <br>
 +
Isolating and PCR: DNA from different trees <br>
 +
-> A: american amber <br>
 +
-> B: oak tree <br>
 +
-> C: hazelnut tree <br>
 +
-> D: maple tree <br> <br>
 +
 +
1.Homogenise samples: <br>
 +
-Liquid nitrogen+ H20: mortar<br> 
 +
2.Lyse cellmembrane <br>
 +
-Add 400 µl PL1 -> eppi
 +
+10 µl RNAse, vortex 30 sec. <br>
 +
-Thermoblock: 10min, 65°C <br>
 +
3.Filtrate <br>
 +
-Put nucleosinfilter in collection tube <br>
 +
-Add lysate <br>
 +
-Centrifugate 2min, 11000 rpm <br>
 +
-Flow volume -> eppi <br>
 +
4.Prepare binding: <br>
 +
Add 450µl Pl,  vortex 10 sec. <br>
 +
5.Bind DNA <br>
 +
-Put nucleosincolumn in collection tube <br>
 +
-Add 700 µl sample <br>
 +
-Centrifugate 1min., 11000 rpm <br>
 +
-Pour away flow volume <br>
 +
5.1 purify <br>
 +
-Put nucleosincolumn in collection tube<br> 
 +
-Add 400µl PW1 <br>
 +
-Centrifugate 1min., 11000 rpm <br>
 +
-Pour away flow volume <br>
 +
5.2 purify again<br> 
 +
-Nucleosincolumn in collection tube<br> 
 +
-Add 700 µl PW2 <br>
 +
-Centrifugate 1min., 11000 rpm <br>
 +
-Pour away flow volume<br> 
 +
5.3 purify again <br>
 +
-Put nucleosincolumn in collection tube <br>
 +
-Add 200µl PW2 <br>
 +
-Centrifugate 2min., 11000 rpm <br>
 +
-Pour away flow volume <br>
 +
6.Eluate  <br>
 +
-Nucleosincolumn in sterile eppi <br>
 +
-Add 50 µl PE <br>
 +
-Thermoblock: 5min., 65 °C<br> 
 +
-Centrifugate 1min., 11000 rpm <br>
 +
-Pour away nucleosincolumn  <br>
 +
-Keep eppi <br>
 +
-> taq PCR <br> <br>
 +
Nanodrop plant DNA
 +
<table style="width 100%">
 +
<tr>
 +
<th> oak tree </th>
 +
<th> ng/µl <br>  260/280<br>  260/230 </th>
 +
<th> 17,5<br>  1,36<br>  0,58 </th>
 +
<th> 14,2 <br>  1,36<br>  0,48 </th>
 +
</tr>
 +
<tr>
 +
<th> hazelnut tree </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 12,8 <br>  1,35 <br>  0,45 </th>
 +
<th> 16,1<br> 1,38 <br>  0,75 </th>
 +
</tr>
 +
<tr>
 +
<th> amber tree </th>
 +
<th> ng/µl <br>  260/230 <br>  260/280 </th>
 +
<th> 4,60<br>  1,37 <br>  0,54 </th>
 +
<th> 53,6 <br>  1,23 <br>  0,66 </th>
 +
</tr>
 +
<tr>
 +
<th> maple tree </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 15,88 <br>  0,84 <br>  0,24 </th>
 +
<th> 10,6 <br>  1,08 <br>  0,44 </th>
 +
</tr>
 +
</table>
 +
 
 +
Gelectrophoresis: did not work-> retry PCR<br> <br> 
 +
 +
26.07.2018<br> <br> 
 +
New plates 200 mg/ml -> 200 µl/ml (diluted) <br>
 +
-Ampicillin+lb <br>
 +
-e.coli+vector-> plated on new plates<br> 
 +
-Plasmids incorporated? <br> <br>
 +
 +
27.07.2018 <br> <br>
 +
-LB+CM plates: did not work <br>
 +
-LB+Amp plates: every colony grew <br>
 +
-> 3 clones plated on LB+Amp plates <br> <br>
 +
 +
14.08.2018 <br> <br>
 +
PCR plant DNA <br>
 +
Taq PCR: birch, oak, hazelnut, amber, maple <br>
 +
-42µl MM <br>
 +
-24 µl Primermix<br> 
 +
-12 µl H2O <br>
 +
-2µl template DNA <br>
 +
-Tm°C: 53<br> <br> 
 +
 
 +
PCR backbones psb1c3 and pz9 <br>
 +
-20 µl phusion buffer <br>
 +
-2µl dNTPs  <br>
 +
-5µl FW primer <br>
 +
-5µl RV primer<br> 
 +
-5µl template DNA <br> 
 +
-0,15 µl DMSO <br>
 +
-61,85 µl H2O <br>
 +
-> each  <br> <br>
 +
 
 +
 +
<table style="width 100%">
 +
<tr>
 +
<th>/</th>
 +
<th> psb1c3 </th>
 +
<th> pz9 </th>
 +
</tr>
 +
<tr>
 +
<th> fragment size </th>
 +
<th> 2070 bp </th>
 +
<th> 5175 bp </th>
 +
</tr>
 +
<tr>
 +
<th> Tm°c </th>
 +
<th> 62°C </th>
 +
<th> 62°C </th>
 +
</tr>
 +
<tr>
 +
<th> extensions (s) </th>
 +
<th> 50s </th>
 +
<th> 125s </th>
 +
</tr>
 +
</table>
 +
 
 +
 +
-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo <br>
 +
                                    Psb1c3 ( did not work) -> cut out insert, new <br> <br>
 +
15.08.2018 <br> <br>
 +
Purify pz9& insert <br>
 +
-> PCR cleanup: Nucleo spin and PCR Clean-up <br> <br> 
 +
Nanodrop pz9 and insert<br>
 +
1.<table style="width 100%">  
 +
<tr>
 +
<th> pz9 </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 2,6 <br>  1,34 <br>  0,79 </th>
 +
<th> 2,5 <br>  0,86 <br>  0,59 </th>
 +
</tr>
 +
<tr>
 +
<th> insert </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 22,6 <br>  1,47 <br>  0,45 </th>
 +
<th> 3,0<br> 3,0 <br>  0,17 </th>
 +
</tr>
 +
</table>
 +
2. <table style="width 100%">
 +
<tr>
 +
<th> pz9 <th>
 +
<th> ng/µl <br>  260/280<br>  260/230 </th>
 +
<th> 2,4 <br>  1,56 <br>  0,78 </th>
 +
<th> 1,8 <br>  1,44 <br>  0,79 </th>
 +
</tr>
 +
<tr>
 +
<th> insert </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 2,8 <br>  2,25 <br>  0,16 </th>
 +
<th> 2,3  <br> 2,28 <br>  0,11 </th>
 +
</tr>
 +
</table><br> <br>
 +
 +
 +
Pz9: new PCR: 4x50 µl <br>
 +
-50µl hf buffer <br>
 +
-5µl dNTPs <br>
 +
-12,5 µl FW primer <br>
 +
-12,5 µl RV primer<br> 
 +
-12,5 µl template DNA <br>
 +
-7,5 µl DMSO <br>
 +
-2,5 µl phusion DNA ploymerase <br>
 +
-147,5 µl H2O <br>
 +
-> worked, multiple seperated bands, purify and cut out <br> <br>
 +
PCR insert XAN: <br>
 +
-36µl phusion hf buffer <br>
 +
-3,6 µl dNTPs <br>
 +
-9µl FW primer  <br>
 +
-9µl RV primer<br> 
 +
-9µl template DNA<br> 
 +
-5,4 µl DMSO <br>
 +
-1,8 µl phusion DNA ploymerase <br>
 +
-106,2 µl H2O <br>
 +
-Fragment size: 5175 bp <br>
 +
-Tm°C: 62 <br>
 +
-Extension: 125s <br>
 +
-DMSO: 3%<br> 
 +
-> gelectrophoresis: did not work, gradient PCR (60,8-70,8°C) <br> <br>
 +
 +
16.08.2018 <br> <br>
 +
Nanodrop purified pz9 <br>
 +
<table style="width 100%">
 +
<tr>
 +
<th> pz9</th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 66,5 <br>  1,38 <br>  0,94 </th>
 +
<th> 65,7 <br>  1,37 <br>  1,19 </th>
 +
</tr>
 +
</table><br> 
 +
 
 +
Xan insert: gradient pcr (55-62 °C) , gelectrophoresis: cut out 7&8 <br> <br>
 +
 +
DNA from different trees<br> 
 +
Retry protocol (14.08.18)<br> 
 +
-95,0 °C, 15 min <br>
 +
-95,0 °C 20 sec <br>
 +
-60,0 °C 40 sec <br>
 +
-72,0 °C 35 sec <br>
 +
-72,0 °C 1 min <br>
 +
-32 cycles <br> <br>
 +
 +
17.08.2018 <br> <br>
 +
Results of the gelelectophoresis of 16.08.: <br>
 +
-> PCR did not work <br>
 +
Possible new methods: <br>
 +
-New primers <br>
 +
-Synthesis (iGEM) <br>
 +
-Linear PCR (one primer) <br>
 +
-2-step PCR <br>
 +
Linear PCR:
 +
-Like Phusion-PCR, but only with one primer <br>
 +
-A-D fw <br>
 +
-E-H rv <br>
 +
-Gradients: 60,8-70,8 °C <br>
 +
-> Gelelectrophoresis <br> <br>
 +
 +
20.08.2018<br> <br> 
 +
PCR of pSB1C3 (2. try): <br>
 +
Phusion-protocol:<br> 
 +
-Templates: 9x20µl <br>
 +
-DMSO: 3% <br>
 +
-Frag.-size: 2070 bp<br> 
 +
-Tm °C: 56-68 °C <br>
 +
-Extension: 45 s <br>
 +
-> Gelelectrophoresis <br>
 +
Results:<br> 
 +
Too many bands: biggest at ca. 3000 bp <br>
 +
-> probably still insert (mCherry) inside the vector <br>
 +
-> possible explanations: primers attached wrong or not at all <br>
 +
-> possible solution: restriction enzymes to cut out the insert before PCR<br>  <br>
 +
Gelelectrophoresis on plant-DNA:<br> 
 +
Repetition of protocol <br> <br>
 +
 +
21.08.2018 <br> <br>
 +
Psb1c3: <br>
 +
Removing mCerry-insert with restriction enzymes:<br> 
 +
<table style="width 100%">
 +
<tr>
 +
<th> prefix </th>
 +
<th> suffix </th>
 +
<th> buffer </th>
 +
<th> Tm°C </th>
 +
</tr>
 +
<tr>
 +
<th> EcoR I (1) </th>
 +
<th> Pst I (1) </th>
 +
<th> O </th>
 +
<th> 37°C </th>
 +
</tr>
 +
<tr>
 +
<th> Xba I (4) </th>
 +
<th> Pst I (1) </th>
 +
<th> Tango </th>
 +
<th> 37°C </th>
 +
</tr>
 +
<tr>
 +
<th> Not I </th>
 +
<th> / </th>
 +
<th> O </th>
 +
<th> 37°C </th>
 +
</tr>
 +
</table>
 +
<br> 
 +
 
 +
 
 +
 
 +
-4nl DNA (200 µg)<br> 
 +
-2 µl Buffer <br>
 +
-1 µl Enzyme <br>
 +
-12 µl water (except for Xbal+Pst I) <br>
 +
-Inactivation: 20 min at 80 °C <br>
 +
-> 2 pieces: 1. pSB1C3 (2070 bp)<br> 
 +
      2. mCehrry (ca. 711 bp) <br> <br>
 +
Nanodrop results:
 +
 
 +
 +
1.measurement
 +
2.measurement
 +
3.measurement
 +
conclusion
 +
260/280
 +
1.83
 +
1.85
 +
1,8
 +
1.82
 +
260/230
 +
1.76
 +
2.01
 +
1,38
 +
1,5
 +
ng/µl
 +
81,1
 +
46,4
 +
48,3
 +
47,4
 +
 +
22.08.2018
 +
Gelelectrophoresis did not completely run through the gel, but different bands visable
 +
-> repetition of restriction with Xbal+Pst I
 +
-> gelelectrophoresis in 0.8 % agarose-gel
 +
Results: 2 clear bands 
 +
Colony PCR
 +
BBa_K523016 (2085 bp)
 +
Filling 200 µl of water in eppis and piercing the lid
 +
Marking 4 colonies on plate and putting them into the eppis
 +
Cooking eppis in the microwave for 3 minutes
 +
-> PCR with taq-polymerase according to orchid-protocol:
 +
Fw primer: BBa_G00100
 +
Rv primer: BBa_G00101
 +
Tm °C: 50 °C
 +
-> Gelelectrophoresis
 +
</article>
 
</div>
 
</div>
 
</div>
 
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Revision as of 14:00, 16 October 2018

Home
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Description Notebook
Overview Methods Pollen PCR Assays Cloning Strategy
Human Practices Marta Event
Safety Form Biosafety
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Notebook

Wet-Lab Protocol

22.05.2018
Opening pollen with Trypsin
-Resuspending 20µg Trypsin in 200 µl of water
-Splitting solution in 4 eppis with 50 µl each => approxamittly 5 µg of Trypsin in each eppi
-5000 U/mg=Proportionalitycalculation
-50mg of pollenmaterial
-3 active Trypsin-eppis (incubating at 30 °C for 15 minutes)
-Approxamittly 15 µg of Trypsin available
Calculating units:
-1 mg= 5000 U
-1 solution: 15 µg= 0.015 mg= 75 U=> 1:3.3
-2 solutions: 7.5 µg=0.0075 mg= 37 U=> 1:6.6
-3 solutions: 5 µg=0.005 mg= 25 U => 1:10
-Proportionality=weight Trypsin : weight pollen
-Add 50 µg of pollen to 10µl Trypsin and 120µl Ammoniumhydrocarbonate
-Incubating at 37°C over night
=> analyzed with light microscopy on 23.05.: did not work

-Microscopy Birchpollen (100x)
-Microscopy Birchpollen (400x)
-Microscopy Willowpollen (100x)
-Microscopy Willowpollen (400x)
-Microscopy Sprucepollen (100x)
-Microscopy Sprucepollen (400x)

Extraction of pollen
-Putting male infructescence into a electrostaticly loaded plastic tube
-Vortexing tube => pollen stick to walls of the tube
-Extracting pollen
-Analyzing with light microscopy if only one type of pollen is in each tube

23.05.2018

Electromicroscopy

-Prepare samples
-> 1: birchpollen
-> 2: Spruce-trypsin supernatant
-> 3: Spruce-trypsin sediment
-> 4: SPruce and spruce ribolysed

-Dried in etikator
-Evaporated with gold
-DNA-extraction pollen
-50 mg in rybolyser tubes
-Constant shaking in rybolyser (6500*2*30*30)
-> centrifugate for 10 min (1100 rpm)

Results:
-Two layers:
-> over the upper one: white shroud
-> light-brown layer of pollen: abstraction with chemical droppper
-> 400µl lysebuffer, centrifugate 1 min (11000 rpm)
-> DNA-extraction: machery nagel plant NUcleo spin III Kit

Nanodrop Samples from leafes
B ng/µl
260/280
260/230 		22,7
1,68
1,23 		97,2
1,71
0,83
PA ng/µl
260/280
260/230 		3,1
0,97
1,05 		8,9
1,83
0,83
PB ng/µl
260/280
260/230 		30,2
2,34
0,73 		35,1
1,84
0,89
Opening pollen with liquid nitrogen
-Nitrogen and mortar and pestle
-Check: light microscope
Opening pollen with ribolyser

-10,20 & 50 mg pollen and 4oo µl water each in ribolyser tube
-Ribolyser (6500-3*45-30)
->Just 10 mg worked (light microscope)

24.05.2018

Pollensamples evaporate with gold (10-20 nm)
nitrogen ng>/µl
260/280
260/230 		66,6
1,88
1,69 		75,6
1,71
1,33
trypsin ng/µl
260/280
260/230 		418,2
1,72
1,00 		399,2
1,59
0,86

DNA-extraction of pollen
-1: opened with trypsin
-2: opened with liquid nitrogen
-> 1: 400 µl PL1 and 1 pollen and 10µl RNA-se ( centrifugate 1min 11000 rpm)
-> 2: 800 µl PL1 and 2 pollen and 10 µl RNA-se ( centrifugate 2min 11000 rpm)
-> protocol DNA extraction

DNA-extraction from birchleafes
-Protocol
DNA-extraction from spruce needles
-Liquid nitrogen

17.07.2018

Extracted DNA: Nanodrop und PCR
-> 5x Phusion HF buffer: 36 µl
-> 10 mn dNTPs: 3.6 µl
-> Fw primer: 9µl
-> rw primer: 9µl
-> template DNA: 9µl
-> Phusion DNA polymerase: 1.8 µl
-> H2o: 111.6 µl
5 primer mixtures: - SP1
- sp2 V - ec1 --> each with DNA sample and positive and negative control
-ec2
-bet

Results Nanodrop
birch pollen+ nitrogen 8ng/µl
spruce pollen+ nitrogen 10,5 ng/µl
birch leaves+ mortar 11,5 ng/µl
birch leaves+mortar 5,5 ng/µl
birch leaves+ mortar 17,5 ng/µl
B. sub -600µl 5c buffer+ cells from plate
-600 µl in ribolyser tubes
-Ribolyse
-Centrifugate: 5 min, 8000 rpm
-Take supernatant +1ml NaCl
-Filtrate

Nanodrop B. Sub.
Leon ng/µl
260/280
260/230 		292,2
1,97
1,12 		185,7
1,59
0,84
Viviane ng/µl
260/280
260/230 		324,7
1,96
1,16 		329,8
1,96
1,16
Jil ng/µl
260/280
260/230 		192,4
1,98
1,19 		263,7
2,01
1,19
Nanodrop plasmid
Fynn ng/µl
260/280
260/230 		37,7
1,96
7,29 		39,1
1,94
5,95
Elisa ng/µl
260/280
260/230 		84,0
1,93
2,24 		83,3
1,97
1,92
Plasmids DNA-isolation: innuprep plasmid mini kit 2.0
-> protocol 1: step 9: 30µl h2o instead of 50-100µ elution buffer
-> one more time 18.07.

18.07.2018

Nanodrop psb1c3
Jil ng/µl
260/280
260/230 		193,6
1,89
2,84 		192,1
1,89
1,78
Elisa ng/µl
260/280
260/230 		33,4
2,00
43,42 		40,0
2,07
-14,57

PCR B.Sub. (bsub_pelB) 1st try

-PCR Phusion 3 step protocol (without DMSO)
-> fragment size: 1038 bp
-> Tm °C: 62
-> extension: 20s/kb
-Breeding from pz9-plasmids
-Isolated pz9 plasmids+
-> phusion HF buffer: 20µl
-> dNTPs: 5µl
-> fw primer:5µl
-> rv primer: 5µl
-> template DNA: 5µl
-> Phusion polymerase: 1µl
-> h20: 62µl
-> fragment size: ca. 5200 bp
-> Tm°C: 59
-> extension: 2:40

Nanodrop Xan.
Ben ng/µl
260/280
260/230 		109,6
1,99
1,21 		122,1
1,93
1,23
Simon ng/µl
260/280
260/230		 109,6
1,96
1,1 		111,4
1,98
1,15


PCR XAN
-1. primer Xan 1+2
-2. primer Xan 3+4
-As breeding pz9
-> fragment size: 745 bp
-> Tm°C: 62
-> 3µl DMSO
-> extension: 30s

PCR B.Sub. 3rd try
-Taq-polymerase
-> long size: 1038 bp
-> Tm°C: 59-70
-> DMS0: 3%

2nd isolation of chromosomal DNA from b.sub. for transformation (standard transformation protocol)
-BBa_JO4450 Trafo
-Promega standard transformation protocol
-Kit 7, 23 O (iGEM)
-50 µl plated
-Centrifugate: 2 min, 5000rpm
-Pellet resuspended, supernatant plated

20.07.2018

Nanodrop bacillus subtilis
B.Sub. 1 ng/µl
260/280
260/230 		93,7
1,71
0,68 		90,8
1,81
0,66
B.Sub. 2 ng/µl
260/280
260/230 		178,2
1,74
0,64 		176,6
1,72
0,66

Nanodrop xanthomonas
Xan.th> ng/µl
260/280
260/230 		35,2
1,92
1,76 		33,1
1,83
1,51
Xan.2 ng/µl
260/280
260/230 		40,4
1,84
1,81 		40,2
1,86
1,80


Gelelectrophoresis psb1c3
-1: 1-4 : E, 5-7: J (kept Dna)
-2: 1-3: E, 4-7: J, 8: size standard (new isolated DNA)

PCR Fynn B.sub. 2nd try
->60,8-70,2 °C

24.07.2018

PCR pz9
-MM: 36µl
-H2O: 117µl
-Primer: 9µl
-Template DNA: 9µl
-> gradient PCR: 62,1-68,9 °C

PCR B.Sub. 4th try
-Taq PCR, new DNA
-> approaches : 2x8
-Frag size: 1038 bp
-Tm°C: 60-70
-DMSO: 3%

PCR Xan. Ben&Simon
-> Phusion: protocol
-Frag size: 700, 1200
-Tm°C: 61,5

Plating trafos
-50µl plated
-Sample centrifugated 3min, 4000 rpm
-Supernatant removed
-Resuspend pellet
-Plate

Isolating and PCR: DNA from different trees
-> A: american amber
-> B: oak tree
-> C: hazelnut tree
-> D: maple tree

1.Homogenise samples:
-Liquid nitrogen+ H20: mortar
2.Lyse cellmembrane
-Add 400 µl PL1 -> eppi +10 µl RNAse, vortex 30 sec.
-Thermoblock: 10min, 65°C
3.Filtrate
-Put nucleosinfilter in collection tube
-Add lysate
-Centrifugate 2min, 11000 rpm
-Flow volume -> eppi
4.Prepare binding:
Add 450µl Pl, vortex 10 sec.
5.Bind DNA
-Put nucleosincolumn in collection tube
-Add 700 µl sample
-Centrifugate 1min., 11000 rpm
-Pour away flow volume
5.1 purify
-Put nucleosincolumn in collection tube
-Add 400µl PW1
-Centrifugate 1min., 11000 rpm
-Pour away flow volume
5.2 purify again
-Nucleosincolumn in collection tube
-Add 700 µl PW2
-Centrifugate 1min., 11000 rpm
-Pour away flow volume
5.3 purify again
-Put nucleosincolumn in collection tube
-Add 200µl PW2
-Centrifugate 2min., 11000 rpm
-Pour away flow volume
6.Eluate
-Nucleosincolumn in sterile eppi
-Add 50 µl PE
-Thermoblock: 5min., 65 °C
-Centrifugate 1min., 11000 rpm
-Pour away nucleosincolumn
-Keep eppi
-> taq PCR

Nanodrop plant DNA
oak tree ng/µl
260/280
260/230 		17,5
1,36
0,58 		14,2
1,36
0,48
hazelnut tree ng/µl
260/280
260/230 		12,8
1,35
0,45 		16,1
1,38
0,75
amber tree ng/µl
260/230
260/280 		4,60
1,37
0,54 		53,6
1,23
0,66
maple tree ng/µl
260/280
260/230 		15,88
0,84
0,24 		10,6
1,08
0,44
Gelectrophoresis: did not work-> retry PCR

26.07.2018

New plates 200 mg/ml -> 200 µl/ml (diluted)
-Ampicillin+lb
-e.coli+vector-> plated on new plates
-Plasmids incorporated?

27.07.2018

-LB+CM plates: did not work
-LB+Amp plates: every colony grew
-> 3 clones plated on LB+Amp plates

14.08.2018

PCR plant DNA
Taq PCR: birch, oak, hazelnut, amber, maple
-42µl MM
-24 µl Primermix
-12 µl H2O
-2µl template DNA
-Tm°C: 53

PCR backbones psb1c3 and pz9
-20 µl phusion buffer
-2µl dNTPs
-5µl FW primer
-5µl RV primer
-5µl template DNA
-0,15 µl DMSO
-61,85 µl H2O
-> each

/ psb1c3 pz9
fragment size 2070 bp 5175 bp
Tm°c 62°C 62°C
extensions (s) 50s 125s
-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo
Psb1c3 ( did not work) -> cut out insert, new

15.08.2018

Purify pz9& insert
-> PCR cleanup: Nucleo spin and PCR Clean-up

Nanodrop pz9 and insert
1.
pz9 ng/µl
260/280
260/230 		2,6
1,34
0,79 		2,5
0,86
0,59
insert ng/µl
260/280
260/230 		22,6
1,47
0,45 		3,0
3,0
0,17
2.
pz9 ng/µl
260/280
260/230 		2,4
1,56
0,78 		1,8
1,44
0,79
insert ng/µl
260/280
260/230 		2,8
2,25
0,16 		2,3
2,28
0,11


Pz9: new PCR: 4x50 µl
-50µl hf buffer
-5µl dNTPs
-12,5 µl FW primer
-12,5 µl RV primer
-12,5 µl template DNA
-7,5 µl DMSO
-2,5 µl phusion DNA ploymerase
-147,5 µl H2O
-> worked, multiple seperated bands, purify and cut out

PCR insert XAN:
-36µl phusion hf buffer
-3,6 µl dNTPs
-9µl FW primer
-9µl RV primer
-9µl template DNA
-5,4 µl DMSO
-1,8 µl phusion DNA ploymerase
-106,2 µl H2O
-Fragment size: 5175 bp
-Tm°C: 62
-Extension: 125s
-DMSO: 3%
-> gelectrophoresis: did not work, gradient PCR (60,8-70,8°C)

16.08.2018

Nanodrop purified pz9
pz9 ng/µl
260/280
260/230 		66,5
1,38
0,94 		65,7
1,37
1,19

Xan insert: gradient pcr (55-62 °C) , gelectrophoresis: cut out 7&8

DNA from different trees
Retry protocol (14.08.18)
-95,0 °C, 15 min
-95,0 °C 20 sec
-60,0 °C 40 sec
-72,0 °C 35 sec
-72,0 °C 1 min
-32 cycles

17.08.2018

Results of the gelelectophoresis of 16.08.:
-> PCR did not work
Possible new methods:
-New primers
-Synthesis (iGEM)
-Linear PCR (one primer)
-2-step PCR
Linear PCR: -Like Phusion-PCR, but only with one primer
-A-D fw
-E-H rv
-Gradients: 60,8-70,8 °C
-> Gelelectrophoresis

20.08.2018

PCR of pSB1C3 (2. try):
Phusion-protocol:
-Templates: 9x20µl
-DMSO: 3%
-Frag.-size: 2070 bp
-Tm °C: 56-68 °C
-Extension: 45 s
-> Gelelectrophoresis
Results:
Too many bands: biggest at ca. 3000 bp
-> probably still insert (mCherry) inside the vector
-> possible explanations: primers attached wrong or not at all
-> possible solution: restriction enzymes to cut out the insert before PCR

Gelelectrophoresis on plant-DNA:
Repetition of protocol

21.08.2018

Psb1c3:
Removing mCerry-insert with restriction enzymes:
prefix suffix buffer Tm°C
EcoR I (1) Pst I (1) O 37°C
Xba I (4) Pst I (1) Tango 37°C
Not I / O 37°C

-4nl DNA (200 µg)
-2 µl Buffer
-1 µl Enzyme
-12 µl water (except for Xbal+Pst I)
-Inactivation: 20 min at 80 °C
-> 2 pieces: 1. pSB1C3 (2070 bp)
2. mCehrry (ca. 711 bp)

Nanodrop results: 1.measurement 2.measurement 3.measurement conclusion 260/280 1.83 1.85 1,8 1.82 260/230 1.76 2.01 1,38 1,5 ng/µl 81,1 46,4 48,3 47,4 22.08.2018 Gelelectrophoresis did not completely run through the gel, but different bands visable -> repetition of restriction with Xbal+Pst I -> gelelectrophoresis in 0.8 % agarose-gel Results: 2 clear bands Colony PCR BBa_K523016 (2085 bp) Filling 200 µl of water in eppis and piercing the lid Marking 4 colonies on plate and putting them into the eppis Cooking eppis in the microwave for 3 minutes -> PCR with taq-polymerase according to orchid-protocol: Fw primer: BBa_G00100 Rv primer: BBa_G00101 Tm °C: 50 °C -> Gelelectrophoresis