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 |
-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 |
-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 |
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 |
-> 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 |