Difference between revisions of "Team:Rheda Bielefeld/Notebook"

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<img src="https://static.igem.org/mediawiki/2018/b/b0/T--Rheda_Bielefeld--ElektromikPrep.jpg" style="width:100%;height:auto;"></img>Leon and Jil prepearing examples for the electrone microscope
 
<img src="https://static.igem.org/mediawiki/2018/b/b0/T--Rheda_Bielefeld--ElektromikPrep.jpg" style="width:100%;height:auto;"></img>Leon and Jil prepearing examples for the electrone microscope
 
<img src="https://static.igem.org/mediawiki/2018/4/4e/T--Rheda_Bielefeld--BenLab.jpg" style="width:100%;height:auto;"></img>Ben working in the lab
 
<img src="https://static.igem.org/mediawiki/2018/4/4e/T--Rheda_Bielefeld--BenLab.jpg" style="width:100%;height:auto;"></img>Ben working in the lab
<img src="https://static.igem.org/mediawiki/2018/0/06/T--Rheda_Bielefeld--LeonSteri.jpg"style="width:100%;height:auto;"></img> Leon working at the sterile bank
+
<img src="https://static.igem.org/mediawiki/2018/0/06/T--Rheda_Bielefeld--LeonSteri.jpg"style="width:100%;height:auto;"></img> Leon working at the slaminar fume hood
 
<img src="https://static.igem.org/mediawiki/2018/4/42/T--Rheda_Bielefeld--BenSchüttler.jpg" style="width:100%;height:auto;"></img>Ben putting colonies into the incubator</div>
 
<img src="https://static.igem.org/mediawiki/2018/4/42/T--Rheda_Bielefeld--BenSchüttler.jpg" style="width:100%;height:auto;"></img>Ben putting colonies into the incubator</div>
 
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<div class="column right">

Revision as of 19:20, 17 October 2018

Leon and Jil prepearing examples for the electrone microscope Ben working in the lab Leon working at the slaminar fume hood Ben putting colonies into the incubator

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)
-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
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


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
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 2070 bp 5175 bp
Tm°c 62°C 62°C
extensions (s) 50s 125s
-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo
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
2.
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