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)
-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
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
-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
3,1 0,97 1,05
8,9 1,83 0,83
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
1,32
trypsin
418,2
1,72
1,00
399,7
1,59
0,86
DNA-extraction of pollen
1: opened with trypsin
2: opened with liquid nitrogen
µ-> 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
- ec1 --> each with DNA sample and positive and negative control
-ec2
-bet
Results Nanodrop
-> birchpollen + nitrogen: 8ng/µl
-> sprucepollen+ nitrogen: 10.5 ng/µl
-> birchleafes+ mortar: 11.5 ng/µl
-> birchleafes+ mortar: 5.5 ng/µl
-> birchleafes+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
324,7
1,96
1,16
329,8
1,96
1,16
Jil
192,4
1,98
1,19
263,7
2,01
1,19
Nanodrop plasmid
Fynn
Ng/µl
260/280
260/260
37,7
1,96
7,29
39,1
1,94
5,95
Elisa
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
33,4
2,0
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
109,6
1,96
1,15
111,4
1,98
1,15
19.07.2018
PCR B.Sub. 2nd try
PCR Phusion protocol
Different temperatures: 60,8-70,2 °C
DMSO 3%
-> gel electrophoresis
PCR Clean-up Xan. (protocol)
Psb1c3: new isolation (innuprep Plasmid Mini Kit 2.0)
Nanodrop
Jil
Ng/µl
260/280
260/230
10,0
2,77
0,42
9,3
3,24
0,6
Elisa
23,6
2,08
2,31
23,4
2,34
1,2
-> PCR as on 18th july
-> gel electrophoresis 1% agarose
Nanodrop Xan.
Viviane
Ng/µl
260/280
260/230
100,1
1,63
0,58
80,9
1,62
0,45
Ben
68,4
1,37
0,15
27,4
1,59
0,11
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
Fynn
b.sub. 1
Ng/µl
260/280
260/230
93,7
1,71
0,68
90,8
1,81
0,66
b. sub. 2
178,2
1,74
0,64
176,6
1,72
0,66
Nanodrop
Viviane
XAn 1
Ng/µl
260/280
260/230
35,2
1,91
1,76
33,1
1,83
1,51
Xan 2
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)
Gelectrophoresis 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
50πl plated
Semple 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
Homogenise samples:
Liquid nitrogen+ H20: mortar
Lyse cellmembrane
Add 400 µl PL1 -> eppi
+10 µl RNAse, vortex 30 sec.
Thermoblock: 10min, 65°C
Filtrate
Put nucleosinfilter in collection tube
Add lysate
Centrifugate 2min, 11000 rpm
Flow volume -> eppi
Prepare binding
Add 450µl Pl, vortex 10 sec.
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
Cetrifugate 2min., 11000 rpm
Pour away flow volume
?
Nucleosincolumn in sterile eppi
Add 50 µl PE
Thermoblock: 5min., 65 °C
Centrifugate 1min., 11000 rpm
Pour away nucleosincolumn
Keep eppi
-> taq PCR
Nanodrop
DNA oak tree
Ng/πl
260/280
260/230
17,5
1,36
0,52
14,2
1,36
48
DNA hazelnut tree
12,8
1,35
0,45
16,1
1,38
0,75
DNA amber
4,6
1,37
0,54
53,6
1,23
0,66
DNA maple tree
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 (bp)
2070
5175
Tm°C
62
62
Extension (s)
50
125
-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo
Psb1c3 ( did not work) -> cut out insert, new PCR
15.08.2018
Purify pz9& insert
-> PCR cleanup: Nucleo spin and PCR Clean-up
Nanodrop
1.
pz9
Ng/µl
260/280
260/230
2,6
1,34
0,79
2,5
0,86
0,59
insert
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
0,71
insert
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
Xbal (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:
1.measurement
2.measurement
3.measurement
conclusion
260/280
1.83
1.85
1,8
1.82
260/230
1.76
2.01
1,38
1,5
ng/µl
81,1
46,4
48,3
47,4
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
