Team:Rheda Bielefeld/Notebook

Leon working at the flow cabinet 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 electrostatically 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