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− | <h2> Labbook </h2>
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− | <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>
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− |
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− | -5000 U/mg=Proportionalitycalculation <br>
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− |
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− | -50mg of pollenmaterial <br>
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− |
| |
− | -3 active Trypsin-eppis (incubating at 30 °C for 15 minutes) <br>
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− |
| |
− | -Approxamittly 15 µg of Trypsin available <br>
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− |
| |
− | Calculating units: <br>
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− |
| |
− | -1 mg= 5000 U <br>
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− |
| |
− | -1 solution: 15 µg= 0.015 mg= 75 U=> 1:3.3 <br>
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− |
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− | -2 solutions: 7.5 µg=0.0075 mg= 37 U=> 1:6.6 <br>
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− |
| |
− | -3 solutions: 5 µg=0.005 mg= 25 U => 1:10<br>
| |
| | | |
− | -Proportionality=weight Trypsin : weight pollen <br> | + | PCR XAN <br> |
− | | + | -1. primer Xan 1+2 <br> |
− | -Add 50 µg of pollen to 10µl Trypsin and 120µl Ammoniumhydrocarbonate <br> | + | -2. primer Xan 3+4 <br> |
− | | + | -As breeding pz9 <br> |
− | -Incubating at 37°C over night <br> | + | -> fragment size: 745 bp<br> |
− | | + | -> Tm°C: 62 <br> |
− | => analyzed with light microscopy on 23.05.: did not work <br><br>
| + | -> 3µl DMSO <br> |
− | | + | -> extension: 30s <br> <br> |
− | | + | PCR B.Sub. 3rd try <br> |
− | -Microscopy Birchpollen (100x) <br> | + | -Taq-polymerase <br> |
− | | + | -> long size: 1038 bp<br> |
− | -Microscopy Birchpollen (400x) <br> | + | -> Tm°C: 59-70 <br> |
− | | + | -> DMS0: 3% <br> <br> |
− | -Microscopy Willowpollen (100x) <br> | + | 2nd isolation of chromosomal DNA from b.sub. for transformation (standard transformation protocol) <br> |
− | | + | -BBa_JO4450 Trafo <br> |
− | -Microscopy Willowpollen (400x) <br> | + | -Promega standard transformation protocol <br> |
− | | + | -Kit 7, 23 O (iGEM) <br> |
− | -Microscopy Sprucepollen (100x) <br> | + | -50 µl plated <br> |
− | | + | -Centrifugate: 2 min, 5000rpm <br> |
− | -Microscopy Sprucepollen (400x) <br><br> | + | -Pellet resuspended, supernatant plated <br> <br> |
− | | + | |
− | | + | |
| | | |
− | Extraction of pollen <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 |
| | | |
− | -Putting male infructescence into a electrostaticly loaded plastic tube <br>
| + | <table style=" width 100%"> |
| | | |
− | -Vortexing tube => pollen stick to walls of the tube <br>
| + | <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> |
| | | |
− | -Extracting pollen <br>
| |
| | | |
− | -Analyzing with light microscopy if only one type of pollen is in each tube <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> |
| | | |
− | 23.05.2018<br>
| + | PCR Fynn B.sub. 2nd try<br> |
− | <br> | + | ->60,8-70,2 °C <br> <br> |
| | | |
− | Electromicroscopy <br>
| |
− | <br>
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− |
| |
− | -Prepare samples <br>
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− |
| |
− | -> 1: birchpollen <br>
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− |
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− | -> 2: Spruce-trypsin supernatant <br>
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− |
| |
− | -> 3: Spruce-trypsin sediment<br>
| |
| | | |
− | -> 4: SPruce and spruce ribolysed <br><br> | + | 24.07.2018 <br> <br> |
− | | + | PCR pz9 <br> |
− | | + | -MM: 36µl <br> |
− | -Dried in etikator<br> | + | -H2O: 117µl <br> |
| + | -Primer: 9µl <br> |
| + | -Template DNA: 9µl <br> |
| + | -> gradient PCR: 62,1-68,9 °C <br> <br> |
| | | |
− | -Evaporated with gold <br>
| + | PCR B.Sub. 4th try <br> |
− | | + | -Taq PCR, new DNA <br> |
− | -DNA-extraction pollen <br> | + | -> approaches : 2x8 <br> |
− | | + | -Frag size: 1038 bp <br> |
− | -50 mg in rybolyser tubes <br> | + | -Tm°C: 60-70 <br> |
− | | + | -DMSO: 3% <br> <br> |
− | -Constant shaking in rybolyser (6500*2*30*30) <br> | + | |
− | | + | |
− | -> centrifugate for 10 min (1100 rpm)<br><br> | + | |
− | | + | |
| | | |
− | Results: <br>
| + | PCR Xan. Ben&Simon <br> |
− | | + | -> Phusion: protocol <br> |
− | -Two layers:<br> | + | -Frag size: 700, 1200<br> |
| + | -Tm°C: 61,5 <br> <br> |
| | | |
− | -> over the upper one: white shroud <br> | + | Plating trafos<br> |
− | | + | -50µl plated <br> |
− | -> light-brown layer of pollen: abstraction with chemical droppper<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> |
| | | |
− | -> 400µl lysebuffer, centrifugate 1 min (11000 rpm) <br> | + | 1.Homogenise samples: <br> |
− | | + | -Liquid nitrogen+ H20: mortar<br> |
− | -> DNA-extraction: machery nagel plant NUcleo spin III Kit <br><br> | + | 2.Lyse cellmembrane <br> |
− | | + | -Add 400 µl PL1 -> eppi |
− | | + | +10 µl RNAse, vortex 30 sec. <br> |
− | | + | -Thermoblock: 10min, 65°C <br> |
− | Nanodrop
| + | 3.Filtrate <br> |
− | Samples from leafes
| + | -Put nucleosinfilter in collection tube <br> |
− | | + | -Add lysate <br> |
− | <table style= "width 100%"> | + | -Centrifugate 2min, 11000 rpm <br> |
− | <tr> | + | -Flow volume -> eppi <br> |
− | <th> B </th> | + | 4.Prepare binding: <br> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | Add 450µl Pl, vortex 10 sec. <br> |
− | <th> 22,7 <br> 1,68 <br> 1,23 </th> | + | 5.Bind DNA <br> |
− | <th> 97,2 <br> 1,71 <br> 0,83 </th> | + | -Put nucleosincolumn in collection tube <br> |
− | </tr> | + | -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> | | <tr> |
− | <th> PA </th> | + | <th> oak tree </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | <th> ng/µl <br> 260/280<br> 260/230 </th> |
− | | + | <th> 17,5<br> 1,36<br> 0,58 </th> |
− | <th> 3,1<br> 0,97 <br> 1,05 </th> | + | <th> 14,2 <br> 1,36<br> 0,48 </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> | | </tr> |
− | </table>
| |
− | Opening pollen with liquid nitrogen <br>
| |
− | -Nitrogen and mortar and pestle <br>
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− | -Check: light microscope <br>
| |
− | Opening pollen with ribolyser <br><br>
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− | -10,20 & 50 mg pollen and 4oo µl water each in ribolyser tube<br>
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− | -Ribolyser (6500-3*45-30) <br>
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− | ->Just 10 mg worked (light microscope) <br><br>
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− |
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− | 24.05.2018 <br><br>
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− | Pollensamples evaporate with gold (10-20 nm)<br>
| |
− | <table style="width 100%">
| |
| <tr> | | <tr> |
− | <th> nitrogen </th> | + | <th> hazelnut tree </th> |
− | <th> ng>/µl <br> 260/280 <br> 260/230 </th> | + | <th> ng/µl <br> 260/280 <br> 260/230 </th> |
− | <th> 66,6 <br> 1,88 <br> 1,69 </th> | + | <th> 12,8 <br> 1,35 <br> 0,45 </th> |
− | <th> 75,6<br> 1,71 <br> 1,33 </th> | + | <th> 16,1<br> 1,38 <br> 0,75 </th> |
| </tr> | | </tr> |
| <tr> | | <tr> |
− | <th> trypsin </th> | + | <th> amber tree </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | <th> ng/µl <br> 260/230 <br> 260/280 </th> |
− | <th> 418,2 <br> 1,72<br> 1,00 </th> | + | <th> 4,60<br> 1,37 <br> 0,54 </th> |
− | <th> 399,2 <br> 1,59 <br> 0,86 </th> | + | <th> 53,6 <br> 1,23 <br> 0,66 </th> |
| </tr> | | </tr> |
− | </table><br>
| |
− |
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− |
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− | DNA-extraction of pollen<br>
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− | -1: opened with trypsin <br>
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− | -2: opened with liquid nitrogen <br>
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− | -> 1: 400 µl PL1 and 1 pollen and 10µl RNA-se ( centrifugate 1min 11000 rpm) <br>
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− | -> 2: 800 µl PL1 and 2 pollen and 10 µl RNA-se ( centrifugate 2min 11000 rpm) <br>
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− | -> protocol DNA extraction <br><br>
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− | DNA-extraction from birchleafes <br>
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− | -Protocol <br>
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− | DNA-extraction from spruce needles <br>
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− | -Liquid nitrogen <br><br>
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− |
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− | 17.07.2018 <br><br>
| |
− | Extracted DNA: Nanodrop und PCR <br>
| |
− | -> 5x Phusion HF buffer: 36 µl <br>
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− | -> 10 mn dNTPs: 3.6 µl <br>
| |
− | -> Fw primer: 9µl <br>
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− | -> rw primer: 9µl <br>
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− | -> template DNA: 9µl <br>
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− | -> Phusion DNA polymerase: 1.8 µl <br>
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− | -> H2o: 111.6 µl <br>
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− | 5 primer mixtures: - SP1 <br>
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− | - sp2 V
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− | - ec1 --> each with DNA sample and positive and negative control <br>
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− | -ec2 <br>
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− | -bet <br><br>
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− | Results Nanodrop <br>
| |
− | <table style ="width 100%">
| |
| <tr> | | <tr> |
− | <th> birch pollen+ nitrogen </th> | + | <th> maple tree </th> |
− | <th> 8ng/µl </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> | | </tr> |
| + | </table> |
| | | |
− | <tr> | + | 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> |
| | | |
− | <th> spruce pollen+ nitrogen </th> | + | 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> |
| | | |
− | <th> 10,5 ng/µl </th>
| + | |
− | | + | <table style="width 100%"> |
− | </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> | | <tr> |
− | <th> birch leaves+ mortar </th> | + | <th>/</th> |
− | <th> 17,5 ng/µl </th> | + | <th> psb1c3 </th> |
| + | <th> pz9 </th> |
| </tr> | | </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> | | <tr> |
− | <th> Leon </th> | + | <th> fragment size </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | <th> 2070 bp </th> |
− | <th> 292,2 <br> 1,97<br> 1,12 </th> | + | <th> 5175 bp </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> |
| <tr> | | <tr> |
− | <th> Jil </th> | + | <th> Tm°c </th> |
− | <th> ng/µl<br> 260/280 <br> 260/230 </th> | + | <th> 62°C </th> |
− | <th> 192,4 <br> 1,98 <br> 1,19 </th> | + | <th> 62°C </th> |
− | <th> 263,7 <br> 2,01 <br> 1,19 </th>
| + | |
| </tr> | | </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> | | <tr> |
− | <th> Elisa</th> | + | <th> extensions (s) </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | <th> 50s </th> |
− | <th> 84,0 <br> 1,93<br> 2,24 </th> | + | <th> 125s </th> |
− | <th> 83,3 <br> 1,97 <br> 1,92 </th>
| + | |
| </tr> | | </tr> |
| </table> | | </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>
| + | -> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo <br> |
− | Nanodrop psb1c3 <br>
| + | Psb1c3 ( did not work) -> cut out insert, new <br> <br> |
− | | + | 15.08.2018 <br> <br> |
− | <table style=" width 100%"> | + | 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> | | <tr> |
− | <th> Jil </th> | + | <th> pz9 </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | <th> ng/µl <br> 260/280 <br> 260/230 </th> |
− | <th> 193,6 <br> 1,89 <br> 2,84 </th> | + | <th> 2,6 <br> 1,34 <br> 0,79 </th> |
− | <th> 192,1<br> 1,89 <br> 1,78 </th> | + | <th> 2,5 <br> 0,86 <br> 0,59 </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> | | </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> | | <tr> |
− | <th> Ben </th> | + | <th> insert </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th> | + | <th> ng/µl <br> 260/280 <br> 260/230 </th> |
− | <th> 109,6 <br> 1,99 <br> 1,21 </th> | + | <th> 22,6 <br> 1,47 <br> 0,45 </th> |
− | <th> 122,1 <br> 1,93 <br> 1,23 </th> | + | <th> 3,0<br> 3,0 <br> 0,17 </th> |
| </tr> | | </tr> |
| + | </table> |
| + | 2. <table style="width 100%"> |
| <tr> | | <tr> |
− | <th> Simon </th> | + | <th> pz9 <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> <br> <br>
| + | |
− | 19.07.2018 <br> <br>
| + | |
− | PCR B.Sub. 2nd try<br>
| + | |
− | -PCR Phusion protocol <br>
| + | |
− | -Different temperatures: 60,8-70,2 °C <br>
| + | |
− | -DMSO 3% <br>
| + | |
− | -> gel electrophoresis<br> <br>
| + | |
− | PCR Clean-up Xan. (protocol) <br>
| + | |
− | -Psb1c3: new isolation (innuprep Plasmid Mini Kit 2.0) <br> <br>
| + | |
− | Nanodrop
| + | |
− | <table style="width 100%">
| + | |
− | <tr>
| + | |
− | <th> Jil </th>
| + | |
| <th> ng/µl <br> 260/280<br> 260/230 </th> | | <th> ng/µl <br> 260/280<br> 260/230 </th> |
− | <th> 10,0 <br> 2,77<br> 0,42 </th> | + | <th> 2,4 <br> 1,56 <br> 0,78 </th> |
− | <th> 9,30 <br> 3,24 <br> 0,60 </th>
| + | <th> 1,8 <br> 1,44 <br> 0,79 </th> |
− | </tr>
| + | |
− | <tr>
| + | |
− | <th> Elisa </th>
| + | |
− | <th> ng/µl <br> 260/280 <br> 260/230 </th>
| + | |
− | <th> 23,6<br> 2,08 <br> 2,31 </th>
| + | |
− | <th> 23,4 <br> 2,34 <br> 1,20 </th>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | -> PCR as on 18th july <br>
| + | |
− | -> gel electrophoresis 1% agarose <br> <br>
| + | |
− | | + | |
− | Nanodrop Xan.
| + | |
− | | + | |
− | <table style="width 100% ">
| + | |
− | <tr>
| + | |
− | <th> Viviane </th>
| + | |
− | <th> ng/µl <br> 260/280<br> 260/230 </th>
| + | |
− | <th> 100,1 <br> 1,63 <br> 0,58 </th>
| + | |
− | <th> 80,9 <br> 1,62 <br> 0,45 </th> | + | |
| </tr> | | </tr> |
| <tr> | | <tr> |
− | <th> Ben </th> | + | <th> insert </th> |
− | <th> ng/µl <br> 260/280 <br> 260/230 <th> | + | <th> ng/µl <br> 260/280 <br> 260/230 </th> |
− | <th> 68,4 <br> 1,37 <br> 0,15 </th> | + | <th> 2,8 <br> 2,25 <br> 0,16 </th> |
− | <th> 27,4 <br> 1,59 <br> 0,11 </th> | + | <th> 2,3 <br> 2,28 <br> 0,11 </th> |
| </tr> | | </tr> |
− | </table> | + | </table><br> <br> |
| | | |
− | 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
| + | Pz9: new PCR: 4x50 µl <br> |
− | Nanodrop
| + | -50µl hf buffer <br> |
− | Fynn
| + | -5µl dNTPs <br> |
− | | + | -12,5 µl FW primer <br> |
− | b.sub. 1
| + | -12,5 µl RV primer<br> |
− | Ng/µl
| + | -12,5 µl template DNA <br> |
− | 260/280
| + | -7,5 µl DMSO <br> |
− | 260/230
| + | -2,5 µl phusion DNA ploymerase <br> |
− | 93,7
| + | -147,5 µl H2O <br> |
− | 1,71
| + | -> worked, multiple seperated bands, purify and cut out <br> <br> |
− | 0,68
| + | PCR insert XAN: <br> |
− | 90,8
| + | -36µl phusion hf buffer <br> |
− | 1,81 | + | -3,6 µl dNTPs <br> |
− | 0,66
| + | -9µl FW primer <br> |
− | b. sub. 2
| + | -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> |
| | | |
− | 178,2
| + | 16.08.2018 <br> <br> |
− | 1,74 | + | Nanodrop purified pz9 <br> |
− | 0,64 | + | <table style="width 100%"> |
− | 176,6
| + | <tr> |
− | 1,72 | + | <th> pz9</th> |
− | 0,66
| + | <th> ng/µl <br> 260/280 <br> 260/230 </th> |
− |
| + | <th> 66,5 <br> 1,38 <br> 0,94 </th> |
− | Nanodrop
| + | <th> 65,7 <br> 1,37 <br> 1,19 </th> |
− | Viviane
| + | </tr> |
| + | </table><br> |
| | | |
− | XAn 1
| + | Xan insert: gradient pcr (55-62 °C) , gelectrophoresis: cut out 7&8 <br> <br> |
− | Ng/µl
| + | |
− | 260/280
| + | |
− | 260/230
| + | |
− | 35,2
| + | |
− | 1,91
| + | |
− | 1,76
| + | |
− | 33,1
| + | |
− | 1,83
| + | |
− | 1,51
| + | |
− | Xan 2
| + | |
| | | |
− | 40,4
| + | DNA from different trees<br> |
− | 1,84
| + | Retry protocol (14.08.18)<br> |
− | 1,81
| + | -95,0 °C, 15 min <br> |
− | 40,2
| + | -95,0 °C 20 sec <br> |
− | 1,86 | + | -60,0 °C 40 sec <br> |
− | 1,80
| + | -72,0 °C 35 sec <br> |
| + | -72,0 °C 1 min <br> |
| + | -32 cycles <br> <br> |
| | | |
− | Gelelectrophoresis psb1c3
| + | 17.08.2018 <br> <br> |
− | 1: 1-4 : E, 5-7: J (kept Dna)
| + | Results of the gelelectophoresis of 16.08.: <br> |
− | 2: 1-3: E, 4-7: J, 8: size standard (new isolated DNA) | + | -> 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> |
| | | |
− | Gelectrophoresis Fynn B.sub. 2nd try
| + | 20.08.2018<br> <br> |
− | 60,8-70,2 °C
| + | 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> |
− | 24.07.2018
| + | Psb1c3: <br> |
− | PCR pz9
| + | Removing mCerry-insert with restriction enzymes:<br> |
− | MM: 36µl
| + | <table style="width 100%"> |
− | H2O: 117µl
| + | <tr> |
− | Primer: 9µl
| + | <th> prefix </th> |
− | Template DNA: 9µl
| + | <th> suffix </th> |
− | -> gradient PCR: 62,1-68,9 °C
| + | <th> buffer </th> |
− | | + | <th> Tm°C </th> |
− | PCR B.Sub. 4th try
| + | </tr> |
− | Taq PCR, new DNA
| + | <tr> |
− | -> approaches : 2x8
| + | <th> EcoR I (1) </th> |
− | Frag size: 1038 bp
| + | <th> Pst I (1) </th> |
− | Tm°C: 60-70
| + | <th> O </th> |
− | DMSO: 3%
| + | <th> 37°C </th> |
− |
| + | </tr> |
− | PCR Xan. Ben&SImon
| + | <tr> |
− | -> Phusion: protocol
| + | <th> Xba I (4) </th> |
− | Frag size: 700, 1200
| + | <th> Pst I (1) </th> |
− | Tm°C: 61,5 | + | <th> Tango </th> |
− |
| + | <th> 37°C </th> |
− | Plating trafos
| + | </tr> |
− | 50πl plated
| + | <tr> |
− | Semple centrifugated 3min, 4000 rpm
| + | <th> Not I </th> |
− | Supernatant removed
| + | <th> / </th> |
− | Resuspend pellet
| + | <th> O </th> |
− | Plate
| + | <th> 37°C </th> |
− | Isolating and PCR: DNA from different trees
| + | </tr> |
− | -> A: american amber
| + | </table> |
− | -> B: oak tree
| + | <br> |
− | -> C: hazelnut tree
| + | |
− | -> D: maple tree
| + | |
− |
| + | |
− | Homogenise samples:
| + | |
− | Liquid nitrogen+ H20: mortar
| + | |
− | Lyse cellmembrane
| + | |
− | Add 400 µl PL1 -> eppi
| + | |
− | +10 µl RNAse, vortex 30 sec.
| + | |
− | Thermoblock: 10min, 65°C
| + | |
− | Filtrate
| + | |
− | Put nucleosinfilter in collection tube
| + | |
− | Add lysate
| + | |
− | Centrifugate 2min, 11000 rpm
| + | |
− | Flow volume -> eppi
| + | |
− | Prepare binding
| + | |
− | Add 450µl Pl, vortex 10 sec.
| + | |
− | 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
| + | |
− | Cetrifugate 2min., 11000 rpm
| + | |
− | Pour away flow volume
| + | |
− | ?
| + | |
− | Nucleosincolumn in sterile eppi
| + | |
− | Add 50 µl PE
| + | |
− | Thermoblock: 5min., 65 °C
| + | |
− | Centrifugate 1min., 11000 rpm
| + | |
− | Pour away nucleosincolumn
| + | |
− | Keep eppi
| + | |
− | -> taq PCR
| + | |
− | Nanodrop
| + | |
| | | |
− | DNA oak tree
| |
− | Ng/πl
| |
− | 260/280
| |
− | 260/230
| |
− | 17,5
| |
− | 1,36
| |
− | 0,52
| |
− | 14,2
| |
− | 1,36
| |
− | 48
| |
− | DNA hazelnut tree
| |
− |
| |
− | 12,8
| |
− | 1,35
| |
− | 0,45
| |
− | 16,1
| |
− | 1,38
| |
− | 0,75
| |
− | DNA amber
| |
− |
| |
− | 4,6
| |
− | 1,37
| |
− | 0,54
| |
− | 53,6
| |
− | 1,23
| |
− | 0,66
| |
| | | |
− | DNA maple tree
| |
− |
| |
− | 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
| |
| | | |
− |
| + | -4nl DNA (200 µg)<br> |
− | psb1c3
| + | -2 µl Buffer <br> |
− | pz9
| + | -1 µl Enzyme <br> |
− | Fragment size (bp)
| + | -12 µl water (except for Xbal+Pst I) <br> |
− | 2070
| + | -Inactivation: 20 min at 80 °C <br> |
− | 5175
| + | -> 2 pieces: 1. pSB1C3 (2070 bp)<br> |
− | Tm°C
| + | 2. mCehrry (ca. 711 bp) <br> <br> |
− | 62
| + | |
− | 62
| + | |
− | Extension (s)
| + | |
− | 50
| + | |
− | 125
| + | |
− |
| + | |
− | -> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo | + | |
− | Psb1c3 ( did not work) -> cut out insert, new PCR
| + | |
− | | + | |
− | 15.08.2018
| + | |
− | Purify pz9& insert
| + | |
− | -> PCR cleanup: Nucleo spin and PCR Clean-up | + | |
− | Nanodrop
| + | |
− | 1.
| + | |
− | | + | |
− | pz9
| + | |
− | Ng/µl
| + | |
− | 260/280
| + | |
− | 260/230
| + | |
− | 2,6 | + | |
− | 1,34
| + | |
− | 0,79
| + | |
− | 2,5
| + | |
− | 0,86
| + | |
− | 0,59
| + | |
− | insert
| + | |
− |
| + | |
− | 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
| + | |
− | 0,71
| + | |
− | insert
| + | |
− |
| + | |
− | 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
| + | |
− | Xbal (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: | | Nanodrop results: |
| | | |