Leon and Jil prepearing examples for the electrone microscope
Ben working in the lab
Leon working at the sterile bank
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)
-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