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

 
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20.07.2018 <br> <br>  
 
20.07.2018 <br> <br>  
Nanodrop <br>  
+
Nanodrop bacillus subtilis <br>
Fynn
+
<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
 +
 
 +
<table style=" width 100%">
 +
 
 +
<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>  
 +
 
  
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
+
Gelelectrophoresis psb1c3 <br>
1,84
+
-1: 1-4 : E, 5-7: J (kept Dna) <br>
1,81
+
-2: 1-3: E, 4-7: J, 8: size standard (new isolated DNA)  <br> <br>
40,2  
+
1,86
+
1,80
+
 
   
 
   
Gelelectrophoresis psb1c3
+
PCR Fynn B.sub. 2nd  try<br> 
1: 1-4 : E, 5-7: J (kept Dna)
+
->60,8-70,2 °C <br> <br>
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 <br> <br>
 +
PCR pz9 <br>
 +
-MM: 36µl <br>
 +
-H2O: 117µl <br>
 +
-Primer: 9µl <br>
 +
-Template DNA: 9µl <br>
 +
-> gradient PCR: 62,1-68,9 °C <br> <br>
 
   
 
   
24.07.2018
+
PCR B.Sub. 4th try <br>
PCR pz9
+
-Taq PCR, new DNA <br>
MM: 36µl
+
-> approaches : 2x8 <br>
H2O: 117µl
+
    -Frag size: 1038 bp <br>
Primer: 9µl
+
    -Tm°C: 60-70 <br>  
Template DNA: 9µl
+
    -DMSO: 3% <br> <br>
-> gradient PCR: 62,1-68,9 °C
+
 
   
 
   
PCR B.Sub. 4th try
+
PCR Xan. Ben&Simon <br>
Taq PCR, new DNA
+
-> Phusion: protocol <br>
-> approaches : 2x8
+
-Frag size: 700, 1200<br> 
    Frag size: 1038 bp
+
-Tm°C: 61,5 <br> <br>
    Tm°C: 60-70
+
    DMSO: 3%
+
 
   
 
   
PCR Xan. Ben&SImon
+
Plating trafos<br> 
-> Phusion: protocol
+
-50µl plated <br>
Frag size: 700, 1200
+
-Sample centrifugated 3min, 4000 rpm <br>
Tm°C: 61,5
+
-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>
 
   
 
   
Plating trafos
+
1.Homogenise samples: <br>
50πl plated
+
-Liquid nitrogen+ H20: mortar<br> 
Semple centrifugated 3min, 4000 rpm
+
2.Lyse cellmembrane <br>
Supernatant removed
+
  -Add 400 µl PL1 -> eppi  
Resuspend pellet
+
+10 µl RNAse, vortex 30 sec. <br>
Plate 
+
-Thermoblock: 10min, 65°C <br>
Isolating and PCR: DNA from different trees
+
3.Filtrate <br>
-> A: american amber
+
-Put nucleosinfilter in collection tube <br>
-> B: oak tree
+
-Add lysate <br>
-> C: hazelnut tree
+
-Centrifugate 2min, 11000 rpm <br>
-> D: maple tree
+
-Flow volume -> eppi <br>
+
4.Prepare binding: <br>
Homogenise samples:  
+
Add 450µl Pl,  vortex 10 sec. <br>
Liquid nitrogen+ H20: mortar  
+
5.Bind DNA <br>
Lyse cellmembrane  
+
-Put nucleosincolumn in collection tube <br>
  Add 400 µl PL1 -> eppi  
+
-Add 700 µl sample <br>
+10 µl RNAse, vortex 30 sec.  
+
-Centrifugate 1min., 11000 rpm <br>
Thermoblock: 10min, 65°C  
+
-Pour away flow volume <br>
Filtrate
+
5.1 purify <br>
Put nucleosinfilter in collection tube  
+
-Put nucleosincolumn in collection tube<br> 
Add lysate  
+
-Add 400µl PW1 <br>
Centrifugate 2min, 11000 rpm  
+
-Centrifugate 1min., 11000 rpm <br>
Flow volume -> eppi  
+
-Pour away flow volume <br>
Prepare binding  
+
5.2 purify again<br> 
Add 450µl Pl,  vortex 10 sec.  
+
-Nucleosincolumn in collection tube<br> 
Bind DNA  
+
-Add 700 µl PW2 <br>
Put nucleosincolumn in collection tube  
+
-Centrifugate 1min., 11000 rpm <br>
Add 700 µl sample  
+
-Pour away flow volume<br> 
Centrifugate 1min., 11000 rpm  
+
5.3 purify again <br>
Pour away flow volume  
+
-Put nucleosincolumn in collection tube <br>
5.1 purify  
+
-Add 200µl PW2 <br>
Put nucleosincolumn in collection tube  
+
-Centrifugate 2min., 11000 rpm <br>
Add 400µl PW1  
+
-Pour away flow volume <br>
Centrifugate 1min., 11000 rpm  
+
6.Eluate  <br>
Pour away flow volume  
+
-Nucleosincolumn in sterile eppi <br>
5.2 purify again  
+
-Add 50 µl PE <br>
Nucleosincolumn in collection tube  
+
-Thermoblock: 5min., 65 °C<br> 
Add 700 µl PW2  
+
-Centrifugate 1min., 11000 rpm <br>
Centrifugate 1min., 11000 rpm  
+
-Pour away nucleosincolumn  <br>
Pour away flow volume  
+
-Keep eppi <br>
5.3 purify again  
+
-> taq PCR <br> <br>
Put nucleosincolumn in collection tube  
+
Nanodrop plant DNA
Add 200µl PW2  
+
<table style="width 100%">
Cetrifugate 2min., 11000 rpm  
+
<tr>
Pour away flow volume  
+
<th> oak tree </th>
?
+
<th> ng/µl <br> 260/280<br>  260/230 </th>
Nucleosincolumn in sterile eppi  
+
<th> 17,5<br>  1,36<br>  0,58 </th>
Add 50 µl PE  
+
<th> 14,2 <br>  1,36<br>  0,48 </th>
Thermoblock: 5min., 65 °C  
+
</tr>
Centrifugate 1min., 11000 rpm  
+
<tr>
Pour away nucleosincolumn   
+
<th> hazelnut tree </th>
Keep eppi  
+
<th> ng/µl <br>  260/280 <br>  260/230 </th>
-> taq PCR  
+
<th> 12,8 <br>  1,35 <br>  0,45 </th>
Nanodrop   
+
<th> 16,1<br> 1,38 <br>  0,75 </th>
 +
</tr>
 +
<tr>
 +
<th> amber tree </th>
 +
<th> ng/µl <br>  260/230 <br>  260/280 </th>
 +
<th> 4,60<br>  1,37 <br>  0,54 </th>
 +
<th> 53,6 <br>  1,23 <br>  0,66 </th>
 +
</tr>
 +
<tr>
 +
<th> maple tree </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>
  
DNA oak tree
+
Gelectrophoresis: did not work-> retry PCR<br> <br> 
Ng/πl
+
260/280
+
260/230
+
17,5
+
1,36
+
0,52
+
14,2
+
1,36
+
48
+
DNA hazelnut tree
+
 
   
 
   
12,8
+
26.07.2018<br> <br> 
1,35
+
New plates 200 mg/ml -> 200 µl/ml (diluted) <br>
0,45
+
-Ampicillin+lb <br>
16,1
+
-e.coli+vector-> plated on new plates<br> 
1,38
+
-Plasmids incorporated? <br> <br>
0,75
+
DNA amber
+
 
   
 
   
4,6
+
27.07.2018 <br> <br>  
1,37
+
-LB+CM plates: did not work <br>
0,54
+
-LB+Amp plates: every colony grew <br>
53,6
+
-> 3 clones plated on LB+Amp plates <br> <br>
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  
+
14.08.2018 <br> <br>
PCR plant DNA  
+
PCR plant DNA <br>
Taq PCR: birch, oak, hazelnut, amber, maple  
+
Taq PCR: birch, oak, hazelnut, amber, maple <br>
42µl MM  
+
-42µl MM <br>
24 µl Primermix  
+
-24 µl Primermix<br> 
12 µl H2O  
+
-12 µl H2O <br>
2µl template DNA  
+
-2µl template DNA <br>
Tm°C: 53  
+
-Tm°C: 53<br> <br> 
PCR backbones psb1c3 and pz9  
+
 
20 µl phusion buffer  
+
PCR backbones psb1c3 and pz9 <br>
2µl dNTPs   
+
-20 µl phusion buffer <br>
5µl FW primer  
+
-2µl dNTPs  <br>
5µl RV primer
+
-5µl FW primer <br>
5µl template DNA   
+
-5µl RV primer<br> 
0,15 µl DMSO  
+
-5µl template DNA <br>  
61,85 µl H2O  
+
-0,15 µl DMSO <br>
-> each   
+
-61,85 µl H2O <br>
 +
-> each  <br> <br>
  
 
   
 
   
psb1c3  
+
<table style="width 100%">
pz9  
+
<tr>
Fragment size (bp)
+
<th>/</th>
2070  
+
<th> psb1c3 </th>
5175  
+
<th> pz9 </th>
Tm°C
+
</tr>
62
+
<tr>
62
+
<th> fragment size </th>
Extension (s)
+
<th> 2070 bp </th>
50
+
<th> 5175 bp </th>
125
+
</tr>
+
<tr>
-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo
+
<th> Tm°c </th>
                                    Psb1c3 ( did not work) -> cut out insert, new PCR
+
<th> 62°C </th>
+
<th> 62°C </th>
15.08.2018
+
</tr>
Purify pz9& insert
+
<tr>
-> PCR cleanup: Nucleo spin and PCR Clean-up 
+
<th> extensions (s) </th>
Nanodrop
+
<th> 50s </th>
1.
+
<th> 125s </th>
 +
</tr>
 +
</table>
  
pz9
 
Ng/µl
 
260/280
 
260/230
 
2,6
 
1,34
 
0,79
 
2,5
 
0,86
 
0,59
 
insert
 
 
   
 
   
22,6  
+
-> gelectrophoresis: pz9 (worked) -> Nanodrop, purify, trafo <br>
1,47  
+
                                    Psb1c3 ( did not work) -> cut out insert, new <br> <br>
0,45  
+
15.08.2018 <br> <br>
3,0  
+
Purify pz9& insert <br>
3,0  
+
-> PCR cleanup: Nucleo spin and PCR Clean-up <br> <br> 
0,17  
+
Nanodrop pz9 and insert<br>
 +
1.<table style="width 100%">
 +
<tr>
 +
<th> pz9 </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 2,6 <br>  1,34 <br>  0,79 </th>
 +
<th> 2,5 <br>  0,86 <br>  0,59 </th>
 +
</tr>
 +
<tr>
 +
<th> insert </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 22,6 <br>  1,47 <br>  0,45 </th>
 +
<th> 3,0<br> 3,0 <br>  0,17 </th>
 +
</tr>
 +
</table>
 +
2. <table style="width 100%">
 +
<tr>
 +
<th> pz9 <th>
 +
<th> ng/µl <br>  260/280<br>  260/230 </th>
 +
<th> 2,4 <br>  1,56 <br>  0,78 </th>
 +
<th> 1,8 <br>  1,44 <br>  0,79 </th>
 +
</tr>
 +
<tr>
 +
<th> insert </th>
 +
<th> ng/µl <br>  260/280 <br>  260/230 </th>
 +
<th> 2,8 <br>  2,25 <br>  0,16 </th>
 +
<th> 2,3  <br> 2,28 <br>  0,11 </th>
 +
</tr>
 +
</table><br> <br>
 
   
 
   
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  
+
Pz9: new PCR: 4x50 µl <br>
50µl hf buffer  
+
-50µl hf buffer <br>
5µl dNTPs  
+
-5µl dNTPs <br>
12,5 µl FW primer  
+
-12,5 µl FW primer <br>
12,5 µl RV primer  
+
-12,5 µl RV primer<br> 
12,5 µl template DNA  
+
-12,5 µl template DNA <br>
7,5 µl DMSO  
+
-7,5 µl DMSO <br>
2,5 µl phusion DNA ploymerase  
+
-2,5 µl phusion DNA ploymerase <br>
147,5 µl H2O  
+
-147,5 µl H2O <br>
-> worked, multiple seperated bands, purify and cut out  
+
-> worked, multiple seperated bands, purify and cut out <br> <br>
PCR insert XAN:  
+
PCR insert XAN: <br>
36µl phusion hf buffer  
+
-36µl phusion hf buffer <br>
3,6 µl dNTPs  
+
-3,6 µl dNTPs <br>
9µl FW primer   
+
-9µl FW primer  <br>
9µl RV primer  
+
-9µl RV primer<br> 
9µl template DNA  
+
-9µl template DNA<br> 
5,4 µl DMSO  
+
-5,4 µl DMSO <br>
1,8 µl phusion DNA ploymerase  
+
-1,8 µl phusion DNA ploymerase <br>
106,2 µl H2O  
+
-106,2 µl H2O <br>
Fragment size: 5175 bp  
+
-Fragment size: 5175 bp <br>
Tm°C: 62  
+
-Tm°C: 62 <br>
Extension: 125s  
+
-Extension: 125s <br>
DMSO: 3%  
+
-DMSO: 3%<br> 
-> gelectrophoresis: did not work, gradient PCR (60,8-70,8°C)  
+
-> gelectrophoresis: did not work, gradient PCR (60,8-70,8°C) <br> <br>
 
   
 
   
16.08.2018  
+
16.08.2018 <br> <br>
Nanodrop purified pz9  
+
Nanodrop purified pz9 <br>
 +
<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> 
  
pz9
+
Xan insert: gradient pcr (55-62 °C) , gelectrophoresis: cut out 7&8 <br> <br>
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  
+
DNA from different trees<br> 
Retry protocol (14.08.18)  
+
Retry protocol (14.08.18)<br> 
95,0 °C, 15 min  
+
-95,0 °C, 15 min <br>
95,0 °C 20 sec  
+
-95,0 °C 20 sec <br>
60,0 °C 40 sec  
+
-60,0 °C 40 sec <br>
72,0 °C 35 sec  
+
-72,0 °C 35 sec <br>
72,0 °C 1 min  
+
-72,0 °C 1 min <br>
32 cycles  
+
-32 cycles <br> <br>
 
   
 
   
17.08.2018  
+
17.08.2018 <br> <br>
Results of the gelelectophoresis of 16.08.:  
+
Results of the gelelectophoresis of 16.08.: <br>
PCR did not work  
+
-> PCR did not work <br>
Possible new methods:  
+
Possible new methods: <br>
New primers  
+
-New primers <br>
Synthesis (iGEM)  
+
-Synthesis (iGEM) <br>
Linear PCR (one primer)  
+
-Linear PCR (one primer) <br>
2-step PCR  
+
-2-step PCR <br>
 
Linear PCR:  
 
Linear PCR:  
Like Phusion-PCR, but only with one primer  
+
-Like Phusion-PCR, but only with one primer <br>
A-D fw  
+
-A-D fw <br>
E-H rv  
+
-E-H rv <br>
Gradients: 60,8-70,8 °C  
+
-Gradients: 60,8-70,8 °C <br>
-> Gelelectrophoresis  
+
-> Gelelectrophoresis <br> <br>
 
   
 
   
20.08.2018  
+
20.08.2018<br> <br> 
PCR of pSB1C3 (2. try):  
+
PCR of pSB1C3 (2. try): <br>
Phusion-protocol:  
+
Phusion-protocol:<br> 
Templates: 9x20µl  
+
-Templates: 9x20µl <br>
DMSO: 3%  
+
-DMSO: 3% <br>
Frag.-size: 2070 bp  
+
-Frag.-size: 2070 bp<br> 
Tm °C: 56-68 °C  
+
-Tm °C: 56-68 °C <br>
Extension: 45 s  
+
-Extension: 45 s <br>
-> Gelelectrophoresis  
+
-> Gelelectrophoresis <br>
Results:  
+
Results:<br> 
Too many bands: biggest at ca. 3000 bp  
+
Too many bands: biggest at ca. 3000 bp <br>
-> probably still insert (mCherry) inside the vector  
+
-> probably still insert (mCherry) inside the vector <br>
-> possible explanations: primers attached wrong or not at all  
+
-> possible explanations: primers attached wrong or not at all <br>
-> possible solution: restriction enzymes to cut out the insert before PCR  
+
-> possible solution: restriction enzymes to cut out the insert before PCR<br>  <br>
Gelelectrophoresis on plant-DNA:  
+
Gelelectrophoresis on plant-DNA:<br> 
Repetition of protocol  
+
Repetition of protocol <br> <br>
 
   
 
   
21.08.2018  
+
21.08.2018 <br> <br>
PSB1C3:  
+
Psb1c3: <br>
Removing mCerry-insert with restriction enzymes:  
+
Removing mCerry-insert with restriction enzymes:<br> 
 +
<table style="width 100%">
 +
<tr>
 +
<th> prefix </th>
 +
<th> suffix </th>
 +
<th> buffer </th>
 +
<th> Tm°C </th>
 +
</tr>
 +
<tr>
 +
<th> EcoR I (1) </th>
 +
<th> Pst I (1) </th>
 +
<th> O </th>
 +
<th> 37°C </th>
 +
</tr>
 +
<tr>
 +
<th> Xba I (4) </th>
 +
<th> Pst I (1) </th>
 +
<th> Tango </th>
 +
<th> 37°C </th>
 +
</tr>
 +
<tr>
 +
<th> Not I </th>
 +
<th> / </th>
 +
<th> O </th>
 +
<th> 37°C </th>
 +
</tr>
 +
</table>
 +
<br> 
  
Prefix
+
 
Suffix
+
 
Buffer
+
-4nl DNA (200 µg)<br> 
Tm °C
+
-2 µl Buffer <br>
EcoR I (1)
+
-1 µl Enzyme <br>
Pst I (1)
+
-12 µl water (except for Xbal+Pst I) <br>
O
+
-Inactivation: 20 min at 80 °C <br>
37 °C
+
-> 2 pieces: 1. pSB1C3 (2070 bp)<br> 
Xbal (4)
+
       2. mCehrry (ca. 711 bp) <br> <br>
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:  
 
  Nanodrop results:  
  

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