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22.05.2018 <br>
 
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Revision as of 11:58, 16 October 2018

Home
The Team Our School Bielefeld University Rheda
Description Notebook
Overview Methods Pollen PCR Assays Cloning Strategy
Human Practices Marta Event
Safety Form Biosafety
Attributions Partners

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
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.


19.07.2018

PCR B.Sub. 2nd try
-PCR Phusion protocol
-Different temperatures: 60,8-70,2 °C
-DMSO 3%
-> gel electrophoresis

PCR Clean-up Xan. (protocol)
-Psb1c3: new isolation (innuprep Plasmid Mini Kit 2.0)

Nanodrop
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
Jil ng/µl
260/280
260/230 		10,0
2,77
0,42 		9,30
3,24
0,60
Elisa ng/µl
260/280
260/230 		23,6
2,08
2,31 		23,4
2,34
1,20
-> PCR as on 18th july
-> gel electrophoresis 1% agarose

Nanodrop Xan.
Viviane ng/µl
260/280
260/230 		100,1
1,63
0,58 		80,9
1,62
0,45
Ben ng/µl
260/280
260/230 		68,4
1,37
0,15 		27,4
1,59
0,11
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 Fynn b.sub. 1 Ng/µl 260/280 260/230 93,7 1,71 0,68 90,8 1,81 0,66 b. sub. 2 178,2 1,74 0,64 176,6 1,72 0,66 Nanodrop Viviane XAn 1 Ng/µl 260/280 260/230 35,2 1,91 1,76 33,1 1,83 1,51 Xan 2 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) Gelectrophoresis 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 50πl plated Semple 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 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 psb1c3 pz9 Fragment size (bp) 2070 5175 Tm°C 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: 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