Leon and Jil prepearing examples for the electron microscope
Ben working in the lab
Leon working at the laminar fume hood
Ben putting colonies into the incubator22.05.2018
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)
-Approximately 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 overnight
=> 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 an electrostatic 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 leaves
| 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 |
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 |
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 |
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 |
| 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:
| 1st measurement | ng/µl 260/280 260/230 |
81,1 1,83 1,76 |
|---|---|---|
| 2nd measurement | ng/µl 260/280 260/230 |
46,4 1,85 2,01 |
| 3rd measurement | ng/µl 260/280 260/230 |
48,3 1,8 1,38 |
| conclusion | ng/µl 260/280 260/230 |
47,4 1,82 1,5 |
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