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

Latest revision as of 13:58, 16 October 2018

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)
-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

-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 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

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 leafes

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: 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

@@ Line 11: / Line 11: @@
 <div class="row">
 <div class="column middle">
-<h2> Labbook </h2>
+<h2> Notebook </h2>
 <article> Wet-Lab Protocol <br><br>
@@ Line 325: / Line 325: @@
 <th> 111,4 <br> 1,98 <br> 1,15 </th>
 </tr>
-</table>{{Template:Rheda_Bielefeld/CSS}}
+</table><br> <br>
-{{Template:Rheda_Bielefeld/Basic_NavBar}}
-<html>
-<head>
-</head>
-<body>
-<div class="header">
-<img src="https://static.igem.org/mediawiki/2018/f/f1/T--Rheda_Bielefeld--dq.jpg" style="width:50%;height:auto;"></img>
-</div>
-<div class="row">
-<div class="column middle">
-<h2> Labbook </h2>
-<article> Wet-Lab Protocol <br><br>
-.05.2018 <br>
-Opening pollen with Trypsin <br>
--Resuspending 20µg Trypsin in 200 µl of water <br>
--Splitting solution in 4 eppis with 50 µl each => approxamittly 5 µg of Trypsin in each eppi <br>
--5000 U/mg=Proportionalitycalculation <br>
--50mg of pollenmaterial <br>
--3 active Trypsin-eppis (incubating at 30 °C for 15 minutes) <br>
--Approxamittly 15 µg of Trypsin available <br>
-Calculating units: <br>
--1 mg= 5000 U <br>
--1 solution: 15 µg= 0.015 mg= 75 U=> 1:3.3 <br>
--2 solutions: 7.5 µg=0.0075 mg= 37 U=> 1:6.6 <br>
--3 solutions: 5 µg=0.005 mg= 25 U => 1:10<br>
--Proportionality=weight Trypsin : weight pollen <br>
+PCR XAN <br>
+-1. primer Xan 1+2 <br>
--Add 50 µg of pollen to 10µl  Trypsin and 120µl Ammoniumhydrocarbonate <br>
+-2. primer Xan 3+4 <br>
+-As breeding pz9  <br>
--Incubating at 37°C over night <br>
+-> fragment size: 745 bp<br>
+-> Tm°C: 62 <br>
-=> analyzed with light microscopy on 23.05.: did not work <br><br>
+-> 3µl DMSO <br>
+-> extension: 30s <br> <br>
+PCR B.Sub. 3rd try <br>
--Microscopy Birchpollen (100x) <br>
+-Taq-polymerase  <br>
+-> long size: 1038 bp<br>
--Microscopy Birchpollen (400x) <br>
+-> Tm°C: 59-70 <br>
+-> DMS0: 3% <br> <br>
--Microscopy Willowpollen (100x) <br>
+nd isolation of chromosomal DNA from b.sub. for transformation (standard transformation protocol) <br>
+-BBa_JO4450 Trafo <br>
--Microscopy Willowpollen (400x) <br>
+-Promega standard transformation protocol <br>
+-Kit 7, 23 O (iGEM) <br>
--Microscopy Sprucepollen (100x) <br>
+-50 µl plated <br>
+-Centrifugate: 2 min, 5000rpm <br>
--Microscopy Sprucepollen (400x) <br><br>
+-Pellet resuspended, supernatant plated <br> <br>
-Extraction of pollen <br>
+.07.2018 <br> <br>
+Nanodrop bacillus subtilis <br>
+<table style="width 100%">
+<tr>
+<th> B.Sub. 1</th>
+<th> ng/µl <br>  260/280 <br> 260/230 </th>
+<th> 93,7 <br>  1,71 <br>  0,68 </th>
+<th> 90,8 <br>  1,81 <br>  0,66 </th>
+</tr>
+<tr>
+<th> B.Sub. 2 </th>
+<th> ng/µl <br>  260/280 <br> 260/230 </th>
+<th> 178,2 <br>  1,74 <br>  0,64 </th>
+<th> 176,6 <br>  1,72 <br>  0,66 </th>
+</tr>
+</table>
+<br>
+Nanodrop xanthomonas
--Putting male infructescence into a electrostaticly loaded plastic tube <br>
+<table style=" width 100%">
--Vortexing tube => pollen stick to walls of the tube <br>
+<tr>
+<th> Xan.th>
+<th> ng/µl <br>  260/280<br>  260/230 </th>
+<th> 35,2 <br>  1,92 <br>  1,76 </th>
+<th> 33,1 <br>  1,83 <br>  1,51 </th>
+</tr>
+<tr>
+<th> Xan.2 </th>
+<th> ng/µl<br>  260/280 <br>  260/230 </th>
+<th> 40,4 <br>  1,84 <br>  1,81 </th>
+<th> 40,2 <br>  1,86 <br>  1,80 </th>
+</tr>
+</table> <br> <br>
--Extracting pollen <br>
--Analyzing with light microscopy if only one type of pollen is in each tube <br>
-<br>
+Gelelectrophoresis psb1c3 <br>
+-1: 1-4 : E, 5-7: J (kept Dna) <br>
+-2: 1-3: E, 4-7: J, 8: size standard (new isolated DNA)  <br> <br>
-.05.2018<br>
+PCR Fynn B.sub. 2nd  try<br>
-<br>
+->60,8-70,2 °C <br> <br>
-Electromicroscopy <br>
-<br>
--Prepare samples <br>
--> 1: birchpollen <br>
--> 2: Spruce-trypsin supernatant <br>
--> 3: Spruce-trypsin sediment<br>
--> 4: SPruce and spruce ribolysed <br><br>
+.07.2018 <br> <br>
+ PCR pz9 <br>
+-MM: 36µl <br>
--Dried in etikator<br>
+-H2O: 117µl <br>
+-Primer: 9µl <br>
+-Template DNA: 9µl <br>
+-> gradient PCR: 62,1-68,9 °C <br> <br>
--Evaporated with gold  <br>
+PCR B.Sub. 4th try <br>
+-Taq PCR, new DNA <br>
--DNA-extraction pollen <br>
+-> approaches : 2x8 <br>
+    -Frag size: 1038 bp <br>
--50 mg in rybolyser tubes <br>
+    -Tm°C: 60-70 <br>
+     -DMSO: 3% <br> <br>
--Constant shaking in rybolyser (6500*2*30*30) <br>
--> centrifugate for 10 min (1100 rpm)<br><br>
-Results: <br>
+PCR Xan. Ben&Simon <br>
+-> Phusion: protocol <br>
--Two layers:<br>
+-Frag size: 700, 1200<br>
+-Tm°C: 61,5 <br> <br>
--> over the upper one: white shroud <br>
+Plating trafos<br>
+-50µl plated <br>
-->  light-brown layer of pollen: abstraction with chemical droppper<br>
+-Sample centrifugated 3min, 4000 rpm <br>
+-Supernatant removed <br>
+-Resuspend pellet <br>
+-Plate  <br> <br>
+Isolating and PCR: DNA from different trees <br>
+-> A: american amber <br>
+-> B: oak tree <br>
+-> C: hazelnut tree <br>
+-> D: maple tree <br> <br>
--> 400µl lysebuffer, centrifugate 1 min (11000 rpm)  <br>
+.Homogenise samples: <br>
+-Liquid nitrogen+ H20: mortar<br>
--> DNA-extraction: machery nagel plant NUcleo spin III Kit <br><br>
+.Lyse cellmembrane <br>
+ -Add 400 µl PL1 -> eppi
++10 µl RNAse, vortex 30 sec. <br>
+-Thermoblock: 10min, 65°C <br>
-Nanodrop
+.Filtrate <br>
-Samples from leafes
+-Put nucleosinfilter in collection tube <br>
+-Add lysate <br>
-<table style= "width 100%">
+-Centrifugate 2min, 11000 rpm <br>
-<tr>
+-Flow volume -> eppi <br>
-<th> B </th>
+.Prepare binding: <br>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
+Add 450µl Pl,  vortex 10 sec. <br>
-<th> 22,7 <br> 1,68 <br> 1,23 </th>
+.Bind DNA <br>
-<th> 97,2 <br> 1,71 <br> 0,83 </th>
+-Put nucleosincolumn in collection tube <br>
-</tr>
+-Add 700 µl sample <br>
+-Centrifugate 1min., 11000 rpm <br>
+-Pour away flow volume <br>
+.1 purify <br>
+-Put nucleosincolumn in collection tube<br>
+-Add 400µl PW1 <br>
+-Centrifugate 1min., 11000 rpm <br>
+-Pour away flow volume <br>
+.2 purify again<br>
+-Nucleosincolumn in collection tube<br>
+-Add 700 µl PW2 <br>
+-Centrifugate 1min., 11000 rpm <br>
+-Pour away flow volume<br>
+.3 purify again <br>
+-Put nucleosincolumn in collection tube <br>
+-Add 200µl PW2 <br>
+-Centrifugate 2min., 11000 rpm <br>
+-Pour away flow volume <br>
+.Eluate  <br>
+-Nucleosincolumn in sterile eppi <br>
+-Add 50 µl PE <br>
+-Thermoblock: 5min., 65 °C<br>
+-Centrifugate 1min., 11000 rpm <br>
+-Pour away nucleosincolumn  <br>
+-Keep eppi <br>
+-> taq PCR <br> <br>
+Nanodrop plant DNA
+<table style="width 100%">
 <tr>
-<th> PA </th>
+<th> oak tree </th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
+<th> ng/µl <br>  260/280<br>  260/230 </th>
+<th> 17,5<br>  1,36<br>  0,58 </th>
-<th> 3,1<br> 0,97 <br> 1,05 </th>
+<th> 14,2 <br>  1,36<br>  0,48 </th>
-<th> 8,9 <br> 1,83 <br> 0,83 </th>
-</tr>
-<tr>
-<th> PB </th>
-<th> ng/µl<br> 260/280 <br> 260/230 </th>
-<th> 30,2 <br> 2,34 <br> 0,73 </th>
-<th> 35,1 <br> 1,84 <br> 0,89 </th>
 </tr>
-</table>
-Opening pollen with liquid nitrogen <br>
--Nitrogen and mortar and pestle <br>
--Check: light microscope <br>
-Opening pollen with ribolyser <br><br>
--10,20 & 50 mg pollen and 4oo µl water each in ribolyser tube<br>
--Ribolyser (6500-3*45-30) <br>
-->Just 10 mg worked (light microscope) <br><br>
-.05.2018 <br><br>
-Pollensamples evaporate with gold (10-20 nm)<br>
-<table style="width 100%">
 <tr>
-<th> nitrogen </th>
+<th> hazelnut tree </th>
-<th> ng>/µl <br> 260/280 <br> 260/230 </th>
+<th> ng/µl <br>  260/280 <br>  260/230 </th>
-<th> 66,6 <br> 1,88 <br> 1,69 </th>
+<th> 12,8 <br>  1,35 <br>  0,45 </th>
-<th> 75,6<br> 1,71 <br> 1,33 </th>
+<th> 16,1<br> 1,38 <br>  0,75 </th>
 </tr>
 <tr>
-<th> trypsin </th>
+<th> amber tree </th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
+<th> ng/µl <br>  260/230 <br>  260/280 </th>
-<th> 418,2 <br> 1,72<br> 1,00 </th>
+<th> 4,60<br>  1,37 <br>  0,54 </th>
-<th> 399,2 <br> 1,59 <br> 0,86 </th>
+<th> 53,6 <br>  1,23 <br>  0,66 </th>
 </tr>
-</table><br>
-DNA-extraction of pollen<br>
--1: opened with trypsin <br>
--2: opened with liquid nitrogen <br>
--> 1: 400 µl PL1 and 1 pollen and 10µl RNA-se ( centrifugate 1min 11000 rpm) <br>
--> 2: 800 µl PL1 and 2 pollen and 10 µl RNA-se ( centrifugate 2min 11000 rpm) <br>
--> protocol DNA extraction <br><br>
- DNA-extraction from birchleafes <br>
--Protocol <br>
-DNA-extraction from spruce needles <br>
--Liquid nitrogen <br><br>
-.07.2018 <br><br>
-Extracted DNA: Nanodrop und PCR <br>
--> 5x Phusion HF buffer: 36 µl <br>
--> 10 mn dNTPs: 3.6 µl <br>
--> Fw primer: 9µl <br>
--> rw primer: 9µl <br>
--> template DNA: 9µl <br>
--> Phusion DNA polymerase: 1.8 µl <br>
--> H2o: 111.6 µl <br>
-primer mixtures: - SP1 <br>
-                                         - sp2 V
-                                         - ec1    --> each with DNA sample and positive and negative control <br>
-                                         -ec2 <br>
-                                         -bet  <br><br>
-Results Nanodrop <br>
-<table style ="width 100%">
 <tr>
-<th> birch pollen+ nitrogen </th>
+<th> maple tree </th>
-<th> 8ng/µl </th>
+<th> ng/µl <br>  260/280 <br>  260/230 </th>
+<th> 15,88 <br>  0,84 <br>  0,24 </th>
+<th> 10,6 <br>  1,08 <br>  0,44 </th>
 </tr>
+</table>
-<tr>
+Gelectrophoresis: did not work-> retry PCR<br> <br>
+.07.2018<br> <br>
+New plates 200 mg/ml -> 200 µl/ml (diluted) <br>
+-Ampicillin+lb <br>
+-e.coli+vector-> plated on new plates<br>
+-Plasmids incorporated? <br> <br>
+.07.2018 <br> <br>
+-LB+CM plates: did not work <br>
+-LB+Amp plates: every colony grew <br>
+-> 3 clones plated on LB+Amp plates <br> <br>
+.08.2018 <br> <br>
+PCR plant DNA <br>
+Taq PCR: birch, oak, hazelnut, amber, maple <br>
+-42µl MM <br>
+-24 µl Primermix<br>
+-12 µl H2O <br>
+-2µl template DNA <br>
+-Tm°C: 53<br> <br>
-<th> spruce pollen+ nitrogen </th>
+PCR backbones psb1c3 and pz9 <br>
+-20 µl phusion buffer <br>
+-2µl dNTPs  <br>
+-5µl FW primer <br>
+-5µl RV primer<br>
+-5µl template DNA <br>
+-0,15 µl DMSO <br>
+-61,85 µl H2O <br>
+-> each  <br> <br>
-<th> 10,5 ng/µl </th>
+<table style="width 100%">
-</tr>
-<tr>
-<th> birch leaves+ mortar </th>
-<th> 11,5 ng/µl </th>
-</tr>
-<tr>
-<th> birch leaves+mortar </th>
-<th> 5,5 ng/µl </th>
-</tr>
 <tr>
-<th> birch leaves+ mortar </th>
+<th>/</th>
-<th> 17,5 ng/µl </th>
+<th> psb1c3 </th>
+<th> pz9 </th>
 </tr>
-</table>
-B. sub
--600µl 5c buffer+ cells from plate <br>
--600 µl in ribolyser tubes <br>
--Ribolyse <br>
--Centrifugate: 5 min, 8000 rpm <br>
--Take supernatant
-+1ml NaCl <br>
--Filtrate <br><br>
-Nanodrop B. Sub.
-<table style ="width 100%">
 <tr>
-<th> Leon </th>
+<th> fragment size </th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
+<th> 2070 bp </th>
-<th> 292,2 <br> 1,97<br> 1,12 </th>
+<th> 5175 bp </th>
-<th> 185,7 <br> 1,59 <br> 0,84 </th>
-</tr>
-<tr>
-<th> Viviane </th>
-<th> ng/µl <br> 260/280<br> 260/230 </th>
-<th> 324,7 <br> 1,96 <br> 1,16 </th>
-<th> 329,8 <br> 1,96 <br> 1,16 </th>
 </tr>
 <tr>
-<th> Jil </th>
+<th> Tm°c </th>
-<th> ng/µl<br> 260/280 <br> 260/230 </th>
+<th> 62°C </th>
-<th> 192,4 <br> 1,98 <br> 1,19 </th>
+<th> 62°C </th>
-<th> 263,7 <br> 2,01 <br> 1,19 </th>
 </tr>
-</table>
-Nanodrop plasmid <br>
-<table style= "width 100%">
 <tr>
-<th> Fynn </th>
+<th> extensions (s) </th>
-<th> ng/µl <br> 260/280<br> 260/230 </th>
+<th> 50s </th>
-<th> 37,7 <br> 1,96<br> 7,29 </th>
+<th> 125s </th>
-<th> 39,1 <br> 1,94 <br> 5,95 </th>
-</tr>
-<tr>
-<th> Elisa</th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
-<th> 84,0 <br> 1,93<br> 2,24 </th>
-<th> 83,3 <br> 1,97 <br> 1,92 </th>
 </tr>
 </table>
-Plasmids DNA-isolation: innuprep plasmid mini kit 2.0 <br>
-                                           -> protocol 1: step 9: 30µl h2o instead of 50-100µ elution buffer <br>
--> one more time 18.07. <br><br>
-.07.2018 <br><br>
+-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo <br>
-Nanodrop psb1c3 <br>
+                                     Psb1c3 ( did not work) -> cut out insert, new <br> <br>
+.08.2018 <br> <br>
-<table style=" width 100%">
+Purify pz9& insert <br>
+-> PCR cleanup: Nucleo spin and PCR Clean-up <br> <br>
+Nanodrop pz9 and insert<br>
+.<table style="width 100%">
 <tr>
-<th> Jil </th>
+<th> pz9 </th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
+<th> ng/µl <br>  260/280 <br>  260/230 </th>
-<th> 193,6 <br> 1,89 <br> 2,84 </th>
+<th> 2,6 <br>  1,34 <br>  0,79 </th>
-<th> 192,1<br> 1,89 <br> 1,78 </th>
+<th> 2,5 <br>  0,86 <br>  0,59 </th>
-</tr>
-<tr>
-<th> Elisa </th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
-<th> 33,4 <br> 2,00 <br> 43,42 </th>
-<th> 40,0 <br> 2,07 <br> -14,57 </th>
 </tr>
-</table> <br>
-PCR B.Sub. (bsub_pelB) 1st try <br><br>
--PCR Phusion 3 step protocol (without DMSO) <br>
--> fragment size: 1038 bp <br>
--> Tm °C: 62 <br>
--> extension: 20s/kb <br>
--Breeding from pz9-plasmids <br>
--Isolated pz9 plasmids+ <br>
--> phusion HF buffer: 20µl <br>
--> dNTPs: 5µl <br>
--> fw primer:5µl <br>
--> rv primer: 5µl <br>
--> template DNA: 5µl <br>
--> Phusion polymerase: 1µl<br>
--> h20: 62µl <br>
--> fragment size: ca. 5200 bp <br>
--> Tm°C: 59 <br>
--> extension: 2:40 <br><br>
-Nanodrop Xan. <br>
-<table style = "width 100% ">
 <tr>
-<th> Ben </th>
+<th> insert </th>
-<th> ng/µl <br> 260/280 <br> 260/230 </th>
+<th> ng/µl <br>  260/280 <br>  260/230 </th>
-<th> 109,6 <br> 1,99 <br> 1,21 </th>
+<th> 22,6 <br>  1,47 <br>  0,45 </th>
-<th> 122,1 <br> 1,93 <br> 1,23 </th>
+<th> 3,0<br> 3,0 <br>  0,17 </th>
 </tr>
+</table>
+. <table style="width 100%">
 <tr>
-<th> Simon </th>
+<th> pz9 <th>
-<th> ng/µl <br> 260/280 <br> 260/230</th>
-<th> 109,6 <br> 1,96<br> 1,1 </th>
-<th> 111,4 <br> 1,98 <br> 1,15 </th> <br> <br>
-.07.2018 <br> <br>
-PCR B.Sub. 2nd try<br>
--PCR Phusion protocol <br>
--Different temperatures: 60,8-70,2 °C <br>
--DMSO 3% <br>
--> gel electrophoresis<br> <br>
-PCR Clean-up Xan. (protocol) <br>
--Psb1c3: new isolation (innuprep Plasmid Mini Kit 2.0) <br> <br>
-Nanodrop
-<table style="width 100%">
-<tr>
-<th> Jil </th>
 <th> ng/µl <br>  260/280<br>  260/230 </th>
-<th> 10,0 <br>  2,77<br>  0,42 </th>
+<th> 2,4 <br>  1,56 <br>  0,78 </th>
-<th> 9,30 <br>  3,24 <br>  0,60 </th>
+<th> 1,8 <br>  1,44 <br>  0,79 </th>
 </tr>
 <tr>
-<th> Elisa </th>
+<th> insert </th>
-<th> ng/µl <br>  260/280 <br> 260/230 </th>
+<th> ng/µl <br>  260/280 <br>  260/230 </th>
-<th> 23,6<br>  2,08 <br>  2,31 </th>
+<th> 2,8 <br>  2,25 <br>  0,16 </th>
-<th> 23,4 <br>  2,34 <br>  1,20 </th>
+<th> 2,3  <br> 2,28 <br>  0,11 </th>
 </tr>
-</table>
+</table><br> <br>
- -> PCR as on 18th july <br>
--> gel electrophoresis 1% agarose <br> <br>
-Nanodrop Xan.
-Viviane
-Ng/µl
-/280
-/230
-,1
-,63
-,58
-,9
-,62
-,45
-Ben
-,4
-,37
-,15
-,4
-,59
-,11
-PCR XAN
+Pz9: new PCR: 4x50 µl <br>
-. primer Xan 1+2
+-50µl hf buffer <br>
-. primer Xan 3+4
+-5µl dNTPs <br>
-As breeding pz9
+-12,5 µl FW primer <br>
--> fragment size: 745 bp
+-12,5 µl RV primer<br>
--> Tm°C: 62
+-12,5 µl template DNA <br>
--> 3µl DMSO
+-7,5 µl DMSO <br>
--> extension: 30s
+-2,5 µl phusion DNA ploymerase <br>
-PCR B.Sub. 3rd try
+-147,5 µl H2O <br>
-Taq-polymerase
+-> worked, multiple seperated bands, purify and cut out <br> <br>
--> long size: 1038 bp
+PCR insert XAN: <br>
--> Tm°C: 59-70
+-36µl phusion hf buffer <br>
--> DMS0: 3%
+-3,6 µl dNTPs <br>
-nd isolation of chromosomal DNA from b.sub. for transformation (standard transformation protocol)
+-9µl FW primer  <br>
-BBa_JO4450 Trafo
+-9µl RV primer<br>
-Promega standard transformation protocol
+-9µl template DNA<br>
-Kit 7, 23 O (iGEM)
+-5,4 µl DMSO <br>
-µl plated
+-1,8 µl phusion DNA ploymerase <br>
-Centrifugate: 2 min, 5000rpm
+-106,2 µl H2O <br>
-Pellet resuspended, supernatant plated
+-Fragment size: 5175 bp <br>
+-Tm°C: 62 <br>
+-Extension: 125s <br>
+-DMSO: 3%<br>
+-> gelectrophoresis: did not work, gradient PCR (60,8-70,8°C) <br> <br>
-.07.2018
+.08.2018 <br> <br>
-Nanodrop
+Nanodrop purified pz9 <br>
-Fynn
+<table style="width 100%">
+<tr>
+<th> pz9</th>
+<th> ng/µl <br>  260/280 <br>  260/230 </th>
+<th> 66,5 <br>  1,38 <br>  0,94 </th>
+<th> 65,7 <br>  1,37 <br>  1,19 </th>
+</tr>
+</table><br>
-b.sub. 1
+Xan insert: gradient pcr (55-62 °C) , gelectrophoresis: cut out 7&8 <br> <br>
-Ng/µl
-/280
-/230
-,7
-,71
-,68
-,8
-,81
-,66
-b. sub. 2
-,2
+DNA from different trees<br>
-,74
+Retry protocol (14.08.18)<br>
-,64
+-95,0 °C, 15 min <br>
-,6
+-95,0 °C 20 sec <br>
-,72
+-60,0 °C 40 sec <br>
-,66
+-72,0 °C 35 sec <br>
+-72,0 °C 1 min <br>
+-32 cycles <br> <br>
-Nanodrop
+.08.2018 <br> <br>
-Viviane
+Results of the gelelectophoresis of 16.08.: <br>
+-> PCR did not work <br>
-XAn 1
+Possible new methods: <br>
-Ng/µl
+-New primers <br>
-/280
+-Synthesis (iGEM) <br>
-/230
+-Linear PCR (one primer) <br>
-,2
+-2-step PCR <br>
-,91
+Linear PCR:
-,76
+-Like Phusion-PCR, but only with one primer <br>
-,1
+-A-D fw <br>
-,83
+-E-H rv <br>
-,51
+-Gradients: 60,8-70,8 °C <br>
-Xan 2
+-> Gelelectrophoresis <br> <br>
-,4
+.08.2018<br> <br>
-,84
+PCR of pSB1C3 (2. try): <br>
-,81
+Phusion-protocol:<br>
-,2
+-Templates: 9x20µl <br>
-,86
+-DMSO: 3% <br>
-,80
+-Frag.-size: 2070 bp<br>
+-Tm °C: 56-68 °C <br>
+-Extension: 45 s <br>
+-> Gelelectrophoresis <br>
+Results:<br>
+Too many bands: biggest at ca. 3000 bp <br>
+-> probably still insert (mCherry) inside the vector <br>
+-> possible explanations: primers attached wrong or not at all <br>
+-> possible solution: restriction enzymes to cut out the insert before PCR<br>  <br>
+Gelelectrophoresis on plant-DNA:<br>
+Repetition of protocol <br> <br>
-Gelelectrophoresis psb1c3
+.08.2018 <br> <br>
-: 1-4 : E, 5-7: J (kept Dna)
+Psb1c3: <br>
-: 1-3: E, 4-7: J, 8: size standard (new isolated DNA)
+Removing mCerry-insert with restriction enzymes:<br>
+<table style="width 100%">
-Gelectrophoresis Fynn B.sub. 2nd  try
+<tr>
-,8-70,2 °C
+<th> prefix </th>
+<th> suffix </th>
+<th> buffer </th>
-.07.2018
+<th> Tm°C </th>
- PCR pz9
+</tr>
-MM: 36µl
+<tr>
-H2O: 117µl
+<th> EcoR I (1) </th>
-Primer: 9µl
+<th> Pst I (1) </th>
-Template DNA: 9µl
+<th> O </th>
--> gradient PCR: 62,1-68,9 °C
+<th> 37°C </th>
+</tr>
-PCR B.Sub. 4th try
+<tr>
-Taq PCR, new DNA
+<th> Xba I (4) </th>
--> approaches : 2x8
+<th> Pst I (1) </th>
-    Frag size: 1038 bp
+<th> Tango </th>
-    Tm°C: 60-70
+<th> 37°C </th>
-     DMSO: 3%
+</tr>
+<tr>
-PCR Xan. Ben&SImon
+<th> Not I </th>
--> Phusion: protocol
+<th> / </th>
-Frag size: 700, 1200
+<th> O </th>
-Tm°C: 61,5
+<th> 37°C </th>
+</tr>
-Plating trafos
+</table>
-πl plated
+<br>
-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
-.1 purify
-Put nucleosincolumn in collection tube
-Add 400µl PW1
-Centrifugate 1min., 11000 rpm
-Pour away flow volume
-.2 purify again
-Nucleosincolumn in collection tube
-Add 700 µl PW2
-Centrifugate 1min., 11000 rpm
-Pour away flow volume
-.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
-/280
-/230
-,5
-,36
-,52
-,2
-,36
-DNA hazelnut tree
-,8
-,35
-,45
-,1
-,38
-,75
-DNA amber
-,6
-,37
-,54
-,6
-,23
-,66
-DNA maple tree
-,88
-,84
-,24
-,6
-,08
-,44
-Gelectrophoresis: did not work-> retry PCR
-.07.2018
-New plates 200 mg/ml -> 200 µl/ml (diluted)
-Ampicillin+lb
-e.coli+vector-> plated on new plates
-Plasmids incorporated?
-.07.2018
-LB+CM plates: did not work
-LB+Amp plates: every colony grew
--> 3 clones plated on LB+Amp plates
-.08.2018
-PCR plant DNA
-Taq PCR: birch, oak, hazelnut, amber, maple
-µl MM
-µl Primermix
-µl H2O
-µl template DNA
-Tm°C: 53
-PCR backbones psb1c3 and pz9
-µl phusion buffer
-µl dNTPs
-µl FW primer
-µl RV primer
-µl template DNA
-,15 µl DMSO
-,85 µl H2O
--> each
+-4nl DNA (200 µg)<br>
-psb1c3
+-2 µl Buffer <br>
-pz9
+-1 µl Enzyme <br>
-Fragment size (bp)
+-12 µl water (except for Xbal+Pst I) <br>
+-Inactivation: 20 min at 80 °C <br>
+-> 2 pieces: 1. pSB1C3 (2070 bp)<br>
-Tm°C
+. mCehrry (ca. 711 bp) <br> <br>
-Extension (s)
--> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo
-                                     Psb1c3 ( did not work) -> cut out insert, new PCR
-.08.2018
-Purify pz9& insert
--> PCR cleanup: Nucleo spin and PCR Clean-up
-Nanodrop
-.
-pz9
-Ng/µl
-/280
-/230
-,6
-,34
-,79
-,5
-,86
-,59
-insert
-,6
-,47
-,45
-,0
-,0
-,17
-.
-pz9
-Ng/µl
-/280
-/230
-,4
-,56
-,78
-,8
-,44
-,71
-insert
-,8
-,25
-,16
-,3
-,28
-,11
-Pz9: new PCR: 4x50 µl
-µl hf buffer
-µl dNTPs
-,5 µl FW primer
-,5 µl RV primer
-,5 µl template DNA
-,5 µl DMSO
-,5 µl phusion DNA ploymerase
-,5 µl H2O
--> worked, multiple seperated bands, purify and cut out
-PCR insert XAN:
-µl phusion hf buffer
-,6 µl dNTPs
-µl FW primer
-µl RV primer
-µl template DNA
-,4 µl DMSO
-,8 µl phusion DNA ploymerase
-,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)
-.08.2018
-Nanodrop purified pz9
-pz9
-Ng/µl
-/280
-/230
-,5
-,38
-,94
-,7
-,37
-,19
-Xan insert: gradient pcr (55-62 °C) , gelectrophoresis: cut out 7&8
-DNA from different trees
-Retry protocol (14.08.18)
-,0 °C, 15 min
-,0 °C 20 sec
-,0 °C 40 sec
-,0 °C 35 sec
-,0 °C 1 min
-cycles
-.08.2018
-Results of the gelelectophoresis of 16.08.:
-PCR did not work
-Possible new methods:
-New primers
-Synthesis (iGEM)
-Linear PCR (one primer)
--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
-.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
-.08.2018
-PSB1C3:
-Removing mCerry-insert with restriction enzymes:
-Prefix
-Suffix
-Buffer
-Tm °C
-EcoR I (1)
-Pst I (1)
-O
-°C
-Xbal (4)
-Pst I (1)
-Tango
-°C
-Not I
-O
-°C
-nl DNA (200 µg)
-µl Buffer
-µl Enzyme
-µl water (except for Xbal+Pst I)
-Inactivation: 20 min at 80 °C
--> 2 pieces: 1. pSB1C3 (2070 bp)
-. mCehrry (ca. 711 bp)
   Nanodrop results: