Difference between revisions of "Team:Freiburg/Labjournals/Cellfree/August"
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<li class="level1"><div class="li"> Due to criticism in the meeting for lacking duplicates or triplicates, the experiment was repeated using biological triplicates for each concentration. (=3 own reactions (from mastermix) for each conc., including MgOAc-feeding)</div> | <li class="level1"><div class="li"> Due to criticism in the meeting for lacking duplicates or triplicates, the experiment was repeated using biological triplicates for each concentration. (=3 own reactions (from mastermix) for each conc., including MgOAc-feeding)</div> | ||
</li> | </li> | ||
− | <li class="level1"><div class="li"> | + | <li class="level1"><div class="li"> Plate layout: Every well contains 20µl of the reaction containing named concentration; A-C are for a 1:2 diluted luc-assay, D-F are for 1:4 diluted luc-assay </div> |
</li> | </li> | ||
</ul> | </ul> | ||
− | <div class="table | + | </p> |
+ | <div class="table sectionedit33"><table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
<th class="col0"> </th><th class="col1">5 </th><th class="col2">6 </th><th class="col3">7 </th><th class="col4">8 </th><th class="col5">9 </th><th class="col6">10 </th><th class="col7">11 </th><th class="col8">12 </th> | <th class="col0"> </th><th class="col1">5 </th><th class="col2">6 </th><th class="col3">7 </th><th class="col4">8 </th><th class="col5">9 </th><th class="col6">10 </th><th class="col7">11 </th><th class="col8">12 </th> | ||
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <th class="col0">A </th><td class="col1"> 1 </td><td class="col2"> | + | <th class="col0"> A </th><td class="col1"> 1 µg </td><td class="col2 leftalign"> 1 µg </td><td class="col3"> 1 µg </td><td class="col4"> 2 µg </td><td class="col5"> 2 µg </td><td class="col6"> 2 µg </td><td class="col7"> 3 µg </td><td class="col8">3 µg </td> |
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <th class="col0">B </th><td class="col1"></td><td class="col2"></td><td class="col3"></td><td class="col4"></td><td class="col5"></td><td class="col6"></td><td class="col7"></td><td class="col8"></td> | + | <th class="col0"> B </th><td class="col1"> 3 µg </td><td class="col2">4 µg </td><td class="col3"> 4 µg </td><td class="col4"> 4 µg </td><td class="col5"> 5 µg </td><td class="col6"> 5 µg </td><td class="col7"> 5 µg </td><td class="col8"> neg </td> |
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <th class="col0">C </th><td class="col1"></td><td class="col2"></td><td class="col3"></td><td class="col4"></td><td class="col5"></td><td class="col6"></td><td class="col7"></td><td class="col8"></td> | + | <th class="col0"> C </th><td class="col1"> neg </td><td class="col2"> neg </td><td class="col3"></td><td class="col4"></td><td class="col5"></td><td class="col6"></td><td class="col7"></td><td class="col8"></td> |
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <th class="col0">D </th><td class="col1"></td><td class="col2"></td><td class="col3"></td><td class="col4"></td><td class="col5"></td><td class="col6"></td><td class="col7"></td><td class="col8"></td> | + | <th class="col0"> D </th><td class="col1"> 1 µg </td><td class="col2"> 1 µg </td><td class="col3"> 1 µg </td><td class="col4"> 2 µg </td><td class="col5"> 2 µg </td><td class="col6"> 2 µg </td><td class="col7"> 3 µg </td><td class="col8"> 3 µg </td> |
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <th class="col0">E </th><td class="col1"></td><td class="col2"></td><td class="col3"></td><td class="col4"></td><td class="col5"></td><td class="col6"></td><td class="col7"></td><td class="col8"></td> | + | <th class="col0"> E </th><td class="col1"> 3 µg </td><td class="col2">4 µg </td><td class="col3"> 4 µg </td><td class="col4"> 4 µg </td><td class="col5"> 5 µg </td><td class="col6"> 5 µg </td><td class="col7"> 5 µg </td><td class="col8"> neg </td> |
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <th class="col0">F </th><td class="col1"> | + | <th class="col0"> F </th><td class="col1"> neg </td><td class="col2"> neg </td><td class="col3"></td><td class="col4"></td><td class="col5"></td><td class="col6"></td><td class="col7"></td><td class="col8"></td> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</tr> | </tr> | ||
</table></div> | </table></div> |
Latest revision as of 12:22, 18 September 2015
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Labjournal Cellfree August
Thu, 27.08.2015
PCR to amplify HA_GFP_HisHis for surface immobilization (Ramona)
- GFP was amplified from PCR product aqSK (with the appropriate binding sites; from AG Roth) with an amino-labeled reverse primer (qSK11) and a Cy3-labeled forward primer (qSK13)
Component | Volume [µl] |
---|---|
aqSK | 0.5 |
qSK11 | 2 |
qSK13 | 2.5 |
Phusion High GC Buffer | 10 |
Phusion polymerase | 0.5 |
dNTPs | 2.5 |
ddH2O | 32 |
- 3fold master mix was prepared and divided into 3 PCR tubes
- cycling conditions were chosen according to the lab journal entry from 16.8.15 (Luisa)
- PCR product was cleaned-up with the PCR purification kit from Qiagen
- result: ~60 µl DNA with a concentration of 66.1 ng/µl
- 10 µl of the PCR product were analyzed on a 1 % agarose gel: amplification of GFP was successful (image could not be saved due to the scanner)
Wed, 26.08.2015
Expression of HA-GFP-His6-His6 on NiNTA slide
(Luisa)
- HA-GFP-His6-His6 was expressed in 15 µl reactions
- 2 reactions were performed as usual in a 1.5 ml tube
- 6 reactions were performed by using a mastermix on a NiNTA slide in spots (2 slides with 3 reactions each)
- 4 reactions were performed without DNA on a NiNTA slide in spots (2 slides with 2 reactions each)
- all samples were incubated for 3 hours at 37°C with the slide being covered by parafilm instead of a microsope slide
- after expression the slides will be incubated at 4°C over night and then measured in the iRIf
- for more results please refer to SurChem lab journal
Tue, 25.08.2015
Expression of HA-GFP-His6-His6 on NiNTA slide
(Luisa)
- HA-GFP-His6-His6 was expressed in 15 µl reactions
- 2 reactions were performed as usual in a 1.5 ml tube
- 6 reactions were performed by using a mastermix on a NiNTA slide in spots (2 slides with 3 reactions each)
- 4 reactions were performed without DNA on a NiNTA slide in spots (2 slides with 2 reactions each)
- all samples were incubated for 3 hours at 37°C with the slide being covered by a regular microscope slide
- after expression the slides will be incubated at 4°C over night and then measured in the iRIf
- for more results please refer to SurChem lab journal
Mon, 24.08.2015
Maxi of LUC Plasmid from Promega and of pIG_1104 with Promega Kit
(Sabi)
- Elution in 750 µl of nuclease free water
- Luc: 560 ng/µl
- Tetanus:
Expression of pIG_1104 (tetanus)
(Koko)
- 50µl rx's, 37°C, 2h, 0rpm
- 3x KK + 4,75µl pIG_1104 DNA (=2µg)
- 3x BK + 4,75µl pIG_1104 DNA (=2µg)
- 2x KK + 4,75µl water (neg)
- 2x BK + 4,75µl water (neg)
Sun, 23.08.2015
Concentration of maxi prep of HA-GFP-His6-His6 and pBEStluc
(Luisa)
- 5 columns per sample, elution in 12 µl each, pooled for measurement
Ponceau staining of expression of various GFPs
Sat, 22.08.2015
Western blot of expression of various GFPs
(Luisa)
blots are so bad they have to be repeated anyways. therefore i did not waste any time making the pictures pretty..
Fri, 21.08.2015
Repetition of variation of MgOAc start concentration in triplicates
(Koko)
- finally came round to repeat critizised experiment
- 20 µl rx's
- 800 ng pBESTluc DNA (=2000 ng/50µl)
- variation from 12 mM to 16 mM of MgOAc, as first experiment suggested 14 mM to be the optimum
- 2 h, 37°C, 0 rpm
- adding 20 µl Dilution agent + 50 µl Luciferase Assay reagent (immediately before platereading)
plate layout
1 | 2 | 3 | 4 | 5 | 6 | 7 | |
---|---|---|---|---|---|---|---|
D | 12mM | 13mM | 14mM | 15mM | 16mM | neg.(+Rmix) | neg.(+H2O) |
E | 12mM | 13mM | 14mM | 15mM | 16mM | neg.(+Rmix) | air |
F | 12mM | 13mM | 14mM | 15mM | 16mM | air | air |
Considerations
- choosing smaller rx's was needed because of lack of luciferase assay reagent
- new luciferase assay reagent AGAIN full of fungi, even after sterile filtering and storage at - 20°
- very small pipeting steps between Mg-concentrations: 0,24µl - 0,26µl - 0,28µl - 0,30µl - 0,32µl –> high error quote possible
Results
- results have been very inaccurate/ have high errors and do not confirm the findings of the first experiment.
- i blame the small rx size and therefore very small pipeting volumes for the errors.
- probably another repetition is needed
Maxiprep of HA-GFP-His6-His6 and pBEStluc
(Luisa)
- failed: c= 69 ng/µl and 183 ng/µl in 1 ml
- Repitiotion of experiment from Tuesday
- 50 µl reactions in black/transparent 384 well plate over time (2000 ng DNA/reaction)
- 480/520 nm over time
- after 2 hours spectrum 480/500-700 nm with foil and without
plate | lysate | premix | amino acids | DNA | comment |
---|---|---|---|---|---|
A1 & B1 | Bernhard | Koko | ours | - | negative control |
A2 & B2 | Bernhard | Koko | ours | HA-GFP-His6-His6 | |
A4 & B4 | Bernhard | Koko | ours | His10-GFP-Spy | |
A5 | Bernhard | Koko | ours | GFP iGEM Bielefeld | |
A6 | Bernhard | Koko | ours | His10-GFP-Spy | |
G6 & H6 | Bernhard | promega | ours | His10-GFP-Spy | |
C1 & D1 | Koko | Koko | ours | - | negative control |
C2 & D2 | Koko | Koko | ours | HA-GFP-His6-His6 | |
C4 & D4 | Koko | Koko | ours | His10-GFP-Spy | |
E1 & F1 | Promega | Promega | Promega | - | negative control |
E2 & F2 | Promega | Promega | Promega | HA-GFP-His6-His6 | |
E4 & F4 | Promega | Promega | Promega | His10-GFP-Spy | |
G1 & H1 | Bernhard | Promega | Promega | - | negative control |
G2 & H2 | Bernhard | Promega | Promega | HA-GFP-His6-His6 | |
G4 & H4 | Bernhard | Promega | Promega | His10-GFP-Spy | |
G5 | Bernhard | Promega | Promega | Bielefeld GFP | |
G6 & H6 | Bernhard | Promega | Promega | His10-GFP-Spy |
Thu, 20.08.2015
Comparison of Koko's and Roth's Kit
(Koko)
- Comparing kinetics of GFP expression, with linear and circular templates
- in Koko's Mix: 25µl rx's with 1µg DNA each (=optimimum)
- in Roth's Mix: 25µl rx's with 0,5µg DNA each (=optimimum)
plate layout
1 | 2 | 3 | 4 | |
---|---|---|---|---|
C | Roth negative | Roth mix + 0,5 µg linear | Roth mix + 0,5 µg circular | air |
D | Koko mix + 1 µg circular | Koko mix + 1 µg circular | Koko mix + 1 µg linear | Koko negative |
… | ||||
H | GFP=? | GFP dilution=? |
- overlaying reactions with 25µl of mineral oil (because Normann said so)
- expression in platereader at 37° for 1:30h
- fluorescence measurement every 2min
- in 384 well plate, transparent, square bottom
- added a few measurements after 1:30h because Koko's still looked like it was expressing
RESULTS
- Roths don't know their GFP concentrations, so positive control is kinda worthless. Higher curve was green GFP, so probably very high concentrated; other one „1:9“ of unkown start concentration.
- Roths Kit is expressing their own linear template in an extreme amount very fast, but nothing else:
- circular DNA did not work in Roth's Kit at all
- linear DNA did not work in Koko's Kit
- very slow expression in Koko's Kit. After 1:30 only at less then 1/10th of usual sample emission values (about 8000-9000 in other experiments)
- plasmid DNA is known to express better than linear templates, so why doesn't it work in Roth's Kit at all?
- mineral oil still sucks because its harming the reaction, or is at least quenching emission values.
Cellfree expression on NiNTA
- 3 samples with HA-GFP-His6-His6 DNA (2000 ng in 15 µl) and 3 samples without DNA were spotted on a slide with spotting mask
- 2 slides were prepared as duplicates
- Kokos lysate, premix and own amino acids were used
- spotting pattern:
triplicate | triplicate | triplicate | triplicate | |
---|---|---|---|---|
spot | pos (HA-GFP-His6-His6) | neg (no DNA) | GFP (pos control) | bBSA (neg control) |
spot | pos (HA-GFP-His6-His6) | neg (no DNA) | GFP (pos control) | bBSA (neg control) |
spot | pos (HA-GFP-His6-His6) | neg (no DNA) | GFP (pos control) | bBSA (neg control) |
Miniprep of GFP from iGEM Bielefeld
- elution in 22 µl nuclease free water
- concentration: 389 ng/µl
Maxiprep of HA-GFP-His6-His6 and pBEStluc
- 2 x 250 ml were incubated over night
Kinetic assay for cellfree expression of luciferase
- 3 assays were performed by adding luciferin and dilution reagent (25 µl) to 25 µl reactions in a white 96 well plate
- measurements were taken every two minutes for 20 minutes (A1/B1 = measurement after 0 minutes of expression, A2/B2 = measurement after 2 minutes of expression, …)
- samples were measured for 116 sec
- triplicates A, B & C were measured by storing B and C on ice while measuring A (Premix and DNA were added just before measurement)
- measurement of C was unreasonably noisy
Tue, 18.08.2015
- Western Blot and Dot blot of Expression of today and from first Expression with Vector from Bielefeld
- 25 µl sample was mixed with 7µl 5x SDS-Buffer and heated to 99°C for 15 min
- 25 µl of the samples were pipted on 12,5% SDS Gel
- Gel 1:
^1 ^2 ^3 ^4 ^5 ^6 ^7 ^8 ^9 ^10 ^ 11 ^ 12 ^13 ^ 14 ^ 15^
Marker | E1 | E2 | E3 | f1 | F2 | F3 | G1 | G2 | G3 | H1 | H2 | J3(mistake) | K1 | K2 | K3 | * Gel2: | Marker | L1 | l2 | H3(mistake) | I1 | I2 | I3 | J1 | J2 | L3 (mistake) | PP Bielefeld | PP Bielefeld no DNA | BK Bielefelfd no DNA | BK Bielefeld | F4 |
* Natural Dot Blor was pipetted following the plate layot
- Dot blot with SDS-samples shows the same pipetting mix up like above
- run for 1,5 h at 60 mA
- Blot for 1,5 h at 0,26A
- Blocking in 5% TBS-T with Milkppowder oN @ 4° ( blue box)
- Gel was stained over night with Comassie
Expressing all the GFPs
- 50 µl reactions in black/transparent 384 well plate over time (2000 ng DNA/reaction)
- 480/520 nm over time
- after 2 hours spectrum 480/500-700 nm with foil and without
plate | lysate | premix | amino acids | DNA | comment |
---|---|---|---|---|---|
E1 & F1 | Bernhard | Koko | ours | - | negative control |
E2 & F2 | Bernhard | Koko | ours | HA-GFP-His6-His6 | |
E3 & F3 | Bernhard | Koko | ours | His10-GFP-Spy | |
E4 & F4 | Bernhard | Koko | ours | GFP iGEM Bielefeld | |
G1 & H1 | Koko | Koko | ours | - | negative control |
G2 & H2 | Koko | Koko | ours | HA-GFP-His6-His6 | |
G3 & H3 | Koko | Koko | ours | His10-GFP-Spy | |
G4 & H4 | Bernhard | Promega | Promega | GFP iGEM Bielefeld | |
I1 & J1 | Promega | Promega | Promega | - | negative control |
I2 & J2 | Promega | Promega | Promega | HA-GFP-His6-His6 | |
I3 & J3 | Promega | Promega | Promega | His10-GFP-Spy | |
I4 & J4 | - | - | - | - | air |
K1 & L1 | Bernhard | Promega | Promega | - | negative control |
K2 & L2 | Bernhard | Promega | Promega | HA-GFP-His6-His6 | |
K3 & L3 | Bernhard | Promega | Promega | His10-GFP-Spy | |
K4 & L4 | - | - | - | - | air |
Mon, 17.08.2015
Expression of GFP DNA sample from iGEM Bielefeld (BK, PP)
- tube reactions are measured in plate reader at 488nm/500nm
- a western blot with GFP antibody will be performed
Expression of luciferase in platereader (over time)
Sun, 16.08.2015
Western Blot of expression of pQE-HA-GFP-his-his (Koko)
- gel loading pattern: (12% sds-page, 0,? A, 1:30h)
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
---|---|---|---|---|---|---|---|---|---|
ladder (ProSieve 4-300kDA) | 25µl rx w/ DNA | 25µl rx w/ DNA | 25µl rx, no DNA | 25µl rx, no DNA | GFPhis 500µg/ml | GFPhis 100µg/ml | GFPhis 10µg/ml | GFPhis 1µg/ml | buffer |
- blotting onto PVDF membrane: (0,25A, 1h)
- checking for correct transfer using ponceau staining (was correct)
* antibody staining:
- primary (Rockland anti GFP (goat)) 1:2000, at 4°C o/n
- secondary (Rockland anti Goat HRP (rabbit)) 1:10.000, RT, 1:30h
- blotting and washing procedure after towbin et al. protocol
Results:
- clear bands in Rx w/ DNA at height of GFP-controls at the right size.
- some smaller extra protein visible above GFP band
—> incomplete translational product containing epitope recognized by AB?
Dot Blot of HA-GFP-His6-His6 to test varying plates and coverage over time
(both)
- 3 X washing with TBS-T
- 1 AB anti GFP from Rockland (1:2000) in 2% BSA
- 3x washing with TBS-T
- 2nd AB anti goat
Expression of GFP DNA sample from iGEM Bielefeld (BK, PP)
(Luisa)
on plate | reaction size | performed in | covered with | DNA | premix | lysate | comment |
---|---|---|---|---|---|---|---|
A 4-6 | 50 µl | tube, measured in 384 well plate | - | none | Koko | Bernhard | negative control |
B 4-6 | 50 µl | tube, measured in 384 well plate | - | Bielefeld | Koko | Bernhard | sample |
C 4-6 | 50 µl | tube, measured in 384 well plate | - | none | Promega | Promega | negative control |
D 4-6 | 50 µl | tube, measured in 384 well plate | - | Bielefeld | Promega | Promega | sample |
J 4-6 | 10 µl, 1:32 | tube, measured in 384 well plate | - | none | none | none | expressed GFP, positive control |
K 4-6 | 10 µl, 1:64 | tube, measured in 384 well plate | - | none | none | none | expressed GFP, positive control |
L 4-6 | 10 µl, 1:128 | tube, measured in 384 well plate | - | none | none | none | expressed GFP, positive control |
- reactions are performed over 2 hrs at 37°C in tubes
- incubated for folding at 4°C over night
- tube reactions are measured in plate reader at 488nm/500nm
PCRs for more HA-GFP-His6-His6 with Cy3 and amino label
(Luisa)
- PCRs will be performed in a 100 µl reaction without DMSO in GC buffer
- forward primer is Cy3 labeled, reverse primer has amino label
- program HA-GFP-His6-His6
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
69 | 20 sec |
72 | 60 sec |
72 | 5 |
4 | infinite |
Component | amount [µl] |
---|---|
buffer - Phusion GC | 20 |
dNTPs | 5 |
Primer forward (Cy5 labeled) | 5 |
Primer reverse (amino labeled) | 4 |
Phusion Polymerase | 1 |
Template (pre-purified) <250 ng | 2 |
DMSO | 0 |
H2O to 100µl each | 63 |
Repetition PCRs for more HA-GFP-His6-His6 with Cy3 and amino label
(Luisa)
- PCRs was performed in 2 x 50 µl reaction without DMSO in GC buffer
- forward primer is Cy3 labeled, reverse primer has amino label
- program HA-GFP-His6-His6
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
69 | 20 sec |
72 | 60 sec |
72 | 5 |
4 | infinite |
Component | amount [µl] |
---|---|
buffer - Phusion GC | 10 |
dNTPs | 2.5 |
Primer forward (Cy5 labeled) | 2.5 |
Primer reverse (amino labeled) | 2 |
Phusion Polymerase | 0.5 |
Template (pre-purified) <250 ng | 1 |
DMSO | 0 |
H2O to 100µl each | 31.5 |
→ per tube
- purify one tube for iRIf, hand over unpurified PCR product of other tube
- PCR worked, 2nd tube: 44 ng/µl in 17 µl nuclease free water
Sat, 15.08.2015
- TO DO (Koko)
WesternBlot of the //HA-GFP-His6-His6// from Wed, 12.08
make moar plasmid! (pQEHA-GFP-His6-His6)
Fri, 14.08.2015
Using the //HA-GFP-His6-His6// from Wed, 12.08 for surface immobilization (Koko)
collaboration with SurRIf subgroup.
- spotting onto Ni-NTA surfaces, by hand, using a stencil for uniformity of the spots (made of PDMS)
spot | component | concentration |
---|---|---|
1 | Reaction with HA-GFP-his-his | unknown |
2 | Reaction with HA-GFP-his-his | unknown |
3 | Reaction with HA-GFP-his-his | unknown |
4 | Reaction w/o DNA | unknown |
5 | Reaction w/o DNA | unknown |
6 | Reaction w/o DNA | unknown |
7 | hisGFP lysate | ~1 mg/ml |
8 | hisGFP lysate | ~1 mg/ml |
9 | untagged GFP | ~1 mg/ml |
10 | bBSA | 200 µg/ml |
11 | Max GFP | 0,5mg/ml |
- all spots contain 2µl and have been incubated on the slide over night at 4° in a damp environment (wet towels) to keep them from evaporating.
- afterwards, the PDMS spotting mask is taken off
- the glass slide is N2-dried (using a wafer-gun)
- slide is put onto PDMS-flowchamber, protein-side facing inwards, and softly pressed on.
- slide and flowchamber are then being put into the iRIf-device and connected to the microfluidics.
Flush protocol:
Reagent | # | Flowrate | Priming time | Concentration |
---|---|---|---|---|
Buffer | 1 | 60 | 780 | 1x |
BSA | 2 | 60 | 780 | 10 mg/ml |
Buffer | 3 | 60 | 600 | 1x |
anti HA | 4 | 40 | 450 | 5 ug/ml |
Buffer | 5 | 60 | 300 | 1x |
anti GFP | 6 | 40 | 450 | 3 ug/ml |
Buffer | 7 | 60 | 600 | 1x |
Strep Cy5 | 8 | 40 | 450 | 5 ug/ml |
Buffer | 9 | 60 | 600 | 1x |
Results:
Spot 11 couldnt be evaluated due to air on it. Spot 1-3 show a-HA binding (weak but clear) and good a-GFP binding → successful cellfree expression.
No strepcy5 binding can be detected: Tube to flowchamber pulled off just before that step….
Expression of HA-GFP-His6-His6 to test varying plates and coverage over time
(Luisa)
on plate | reaction size | performed in | covered with | DNA | premix | lysate | comment |
---|---|---|---|---|---|---|---|
A 1-3 | 15 µl | 384 well plate | foil | none | Koko | Bernhard | negative control |
B 1-3 | 15 µl | 384 well plate | foil | HA-GFP-His6-His6 | Koko | Bernhard | sample |
C 1-3 | 15 µl, 1:8 | 384 well plate | foil | none | none | none | expressed GFP, positive control |
D 1-3 | 15 µl | 384 well plate | mineral oil | none | Koko | Bernhard | negative control |
E 1-3 | 15 µl | 384 well plate | mineral oil | HA-GFP-His6-His6 | Koko | Bernhard | sample |
F 1-3 | 15 µl 1:8 | 384 well plate | mineral oil | none | none | none | expressed GFP, positive control |
G 1-3 | 15 µl | tube, measured in plate | nothing, foil | none | Koko | Bernhard | negative control |
H 1-3 | 15 µl | tube, measured in plate | nothing, foil | HA-GFP-His6-His6 | Koko | Bernhard | sample |
I 1-3 | 15 µl, 1:8 | tube, measured in plate | nothing, foil | none | none | none | expressed GFP, positive control |
J 1-3 | 15 µl, 1:16 | tube, measured in plate | nothing, foil | none | none | none | expressed GFP, positive control |
K 1-3 | 15 µl, 1:32 | tube, measured in plate | nothing, foil | none | none | none | expressed GFP, positive control |
L 1-3 | 15 µl, 1:64 | tube, measured in plate | nothing, foil | none | none | none | expressed GFP, positive control |
- reactions are performed over 2 hrs at 37°C
- tube reactions are measured in plate reader after reaction and covered with foil after first measurement
Thu, 13.08.2015
- purification of HA-GFP-His6-His6 with Cy3 and amino label (Luisa)
- concentration: 28 ng/µl
- tubes were covered with aluminium foil to avoid bleaching and handed to surface chemistry
Repetition of variation of DNA-concentration, 1-5µg (Koko)
- Due to criticism in the meeting for lacking duplicates or triplicates, the experiment was repeated using biological triplicates for each concentration. (=3 own reactions (from mastermix) for each conc., including MgOAc-feeding)
- Plate layout: Every well contains 20µl of the reaction containing named concentration; A-C are for a 1:2 diluted luc-assay, D-F are for 1:4 diluted luc-assay
5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
---|---|---|---|---|---|---|---|---|
A | 1 µg | 1 µg | 1 µg | 2 µg | 2 µg | 2 µg | 3 µg | 3 µg |
B | 3 µg | 4 µg | 4 µg | 4 µg | 5 µg | 5 µg | 5 µg | neg |
C | neg | neg | ||||||
D | 1 µg | 1 µg | 1 µg | 2 µg | 2 µg | 2 µg | 3 µg | 3 µg |
E | 3 µg | 4 µg | 4 µg | 4 µg | 5 µg | 5 µg | 5 µg | neg |
F | neg | neg |
- Results
Wed, 12.08.2015
(Koko)
Expression of HA-GFP-His6-His6
- 50µl reaction of complete Koko-system
- using the evaluated best-working DNA concentration of 2 µg (see 08.08.)
- using the evaluated best-working MgOAc-concentration of 14mM (see 10.08)
- 2h, 37°C, 0rpm
planned to use for:
spotting the finished expression onto Ni-NTA slides in collaboration with SurChem subgroup, to check for working immobilization of the tagged GFP
WesternBlot to confirm correct expression
Repetition of MgOAc-variation in Bernhard-Koko system from Mon, 10.08.
- this time using the right concentrations, otherwise exactly the same
Results:
- system completely dead, no expression
- –> Bernhard mix does not, as promised, work best between 15 and 18mM.
- other explanation: our stock of MgOAc is unknowingly slightly higher concentrated than we think it is
PCRs of HA-GFP-His6-His6 with Cy3 and amino label
(Luisa)
- PCRs will be performed in a 50 µl reaction without DMSO in GC buffer
- forward primer is Cy3 labeled, reverse primer has amino label
- program HA-GFP-His6-His6
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
69 | 20 sec |
72 | 60 sec |
72 | 5 |
4 | infinite |
Component | amount [µl] |
---|---|
buffer - Phusion GC | 10 |
dNTPs | 2.5 |
Primer forward (Cy5 labeled) | 2.5 |
Primer reverse (amino labeled) | 2 |
Phusion Polymerase | 0.5 |
Template (pre-purified) <250 ng | 1 |
DMSO | 0 |
H2O to 100µl each | 31.5 |
Tue, 11.08.2015
- Continuation of Western and Dot Blot of Expression of pIG15_105 from yesterday (Sabi)
- 3 x washing with TBS-T
- first antibody: anti-tYFP (Evrogen) (1:5000)
- 3 x washing with TBS-T
- sec. antibody: anti-rabbit 1:20000 (Promega)
- 3x washing with TBS-T
- Signal at the right hight around 61 kDa (ca 27kDa tYFP + 34 kDa Halo)
Mon, 10.08.2015
- Variation of start-concentration of MgOAc (Koko)
- in both Bernhard+Koko and complete Koko for further improvement of reaction conditions
- Bernhard once said his system was optimal in the range of 15-18mM.
- no further feeding of the mix.
- 2h, 37°C, 0rpm
- 4µg of pBESTluc DNA to compare expression via Luciferase assay later on
- first one was not (falsely) adjusted (= 11mM)–> visible maximum
- –> will be repeated (see 12.08.)
Results:
- Dot Blot and Western Blot of Expressionfrom today (105): (Sabi)
- before measuring in platereader: 10 µl of reaction were passed on to SurChem
- 15µl were used for measurement in platereader
- the remaining 25 µl were mixed with 7 µl 5X SDS sample buffer and heated too 99°C for 15 min
- 2µl of the samples were spotted for a Dot Blot on activated PVDF
- After measurement in the platereader 2 µl of the reaction mix that were spotted as well (without SDS)
- for the Western Blot 25µl sample were used
- Pippetting sheme: Marker-no DnA-K/K-K/K+feed-B/K-B/K+feed
- gel run for 1,5 h at 0,03 A but looked slightly strange –> SDS sample buffer might be invested with foreign proteins
- in the biginning gel rumn very slow only on centimeter in an hour. Probably due to the missing second balance glass plate. Glass plate was attached and the run started again
- blotting at 0,14A for 1,5 h with the small blotting apperature
- whole marker was on the membrane and a little bit on the Whatman behind –> no further increase of Ampere
- blocking in TBS-T with 5% milkpowder oN at 4°C
- Continuation of Dot and Western blot from yesterday (104)
- 3x washing with TBS-T
- first anti-body: anti-YFP (1:5000)
- 3x Washing with TBS-T
- sec. Antibody: anti Rabbit (1:20000)
- 3 washing with TBS-T
- clear spots with KK+ /KK- –> However in the platereader B performed better
- only natural spotted expression mix shows clear signal –> perhaps due to differnt amount of proteins, as indicated by the ponceau staining
- two bands at the height of tYFP
expression of pIG15_105 (Prep) (Luisa)
- cellfree expression of pIG15_105 (His-tYFP-Spy) by using 5 µg of DNA with a mix of various lysates, amino acids and premixes
- selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
- fluorescence measurement was performed at an excitation of 488 nm
K = Koko
B = Bernhard
Lysate | Premix | Amino Acids | Feed? | comment |
---|---|---|---|---|
K | K | ours | Mg(OAc)2 | - |
K | K | ours | H2O | - |
B | K | ours | Mg(OAc)2 | - |
B | K | ours | H2O | - |
K | K | ours | H2O | negative control |
Sun, 09.08.2015
- Continuation of Dot Blot of GFP Expression from yesterday (Sabi)
- 3 X washing of membrane with TBS-T
- exposure for 4 min and 8 min
- Dot Blot of Expression YFP from yesterday: (Sabi)
- Dot Blot and Western Blot of Expressionfrom today: (Sabi)
- before measuring in platereader: 10 µl of reaction were passed on to SurChem
- 15µl were used for measurement in platereader
- the remaining 25 µl were mixed with 7 µl 5X SDS sample buffer and heated too 99°C for 15 min
- 2µl of the samples were spotted for a Dot Blot on activated PVDF
- After measurement in the platereader 2 µl of the reaction mix that were spotted as well (without SDS)
- for the Western Blot 25µl samples were used
- Pippetting scheme: Marker-no DnA-K/K-K/K+feed-B/K-B/K+feed
- gel run for 2 h at 0,03 A but looked slightly strange –> SDS sample buffer might be contaminated invested with other proteins
- blotting at 0,13A for 1,5 h
together with teh Dot Blot the membranes were blocked oN in 5% Milkpowder TBS-T
repeat of yesterdays expression with pIG15_104 by using the intended amount of DNA (5 µg) (Luisa)
- cellfree expression of pIG15_104 (His-tYFP-Spy) by using 5 µg of DNA with a mix of various lysates, amino acids and premixes
- selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
- fluorescence measurement was performed at an excitation of 488 nm
K = Koko
B = Bernhard
Lysate | Premix | Amino Acids | Feed? | comment |
---|---|---|---|---|
K | K | ours | Mg(OAc)2 | - |
K | K | ours | H2O | - |
B | K | ours | Mg(OAc)2 | - |
B | K | ours | H2O | - |
K | K | ours | H2O | negative control |
Sat, 08.08.2015
Variation of DNA-concentration from 1-5µg
- 5 expressions and a negative control in 50µl size were carried out in parallel:
- Mastermix for 5 rx:
component | amount from stock in µl |
---|---|
S30 Lysate | 112,5 |
HEPES-KOH ph 7,5 | 13,75 |
DTT | 4,25 |
ATP pH7 | 3 |
CTP | 2 |
GTP | 2 |
UTP | 2 |
Creat.Phos | 20 |
CreatineKin | 0,875 |
PEG4k | 5 |
cAMP | 1,625 |
folinic acid | 8,75 |
tRNA | 5,7 |
pot.glutamat | 18,5 |
MgOAc | 2,75 |
Aminos | 9,375 |
- The Premix for 5 rx w/o lysate adds up to 99,575µl = 19,915µl per 50µl reaction.
- The negative control consists of 22,5µl lysate + 27,5µl water, no DNA.
- Reaction carried out as usual: 2h, 37°C, 0rpm
- Reactions were feeded 0,5µl of 10µM MgOAc every 20min.
DNA variation:
- Expression of pBESTluc plasmid (c=3151,4ng/µl)
DNA | amount from stock in µl |
---|---|
1µg | 0,31 |
2µg | 0,63 |
3µg | 0,95 |
4µg | 1,27 |
5µg | 1,58 |
0µg | 1,00 (H2O) |
- 6,01-7,28 µl of water ad 50µl
- Luciferase Assay to compare expression:
- 20µl of reaction+ 20µl of dilution agent for each reaction
- in 96well plate, white, flatbottom
- adding 50µl of luciferase assay reagent (=luciferin+cofactors) to each well right before measurement
- full spectrum scan (300-700nm) in microplate reader
Results
- highest expression for DNA=2µg
- negative control showed signal higher than 1µg, which suggests errors in pipeting or mix up of data.
- Experiment was repeated with triplicates on 13.08.15
- Dot Blot of GFP of last Expression from yesterday (Sabi)
- on the activated PVDF membrane the 7 samples and a GFP positiv control were spotted
- The membrane was blocked for 1 hour in 5%milkpowder in TBS-T
- 3 x washing with TBS-T
- 2 h incubation with anti GFP (1:2000) (mouse)
- 3 x washing with TBS-T
- incubation oN at 4°C with anti mouse ab
- measurement of the GFP Expression from 7.8.2015 (Sabi)
- Excitation 488 –> Emission 509-700 spectral scanning
- white plates
- remeasurement with black plates and see-through plates
- no big difference between those kinds of plates. however white plaes increase the signal and gives a distorted picture of reality.
- for example were the negative controls over 20000 relative fluorecence units
expression of pIG15_104 (Prep) (Luisa)
- cellfree expression of pIG15_104 (His-tYFP-Spy) by using 0.5 µg of DNA with a mix of various lysates, amino acids and premixes
- selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
- after expression the plate was measured covered with foil and without foil to test wether an expression in the platereader without evaporation of the reaction would be possible
- fluorescence measurement was performed at an excitation of 488 nm
K = Koko
B = Bernhard
Lysate | Premix | Amino Acids | Feed? | comment |
---|---|---|---|---|
K | K | ours | Mg(OAc)2 | - |
K | K | ours | H2O | - |
B | K | ours | Mg(OAc)2 | - |
B | K | ours | H2O | - |
K | K | ours | H2O | negative control |
Fri, 07.08.2015
expression of HA-GFP-His6-His6 (Prep) (Koko)
- cellfree expression of HA-GFP-His6-His6 by using 3,25 µg of DNA with a mix of various lysates, amino acids and premixes
- selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
- fluorescence measurement was performed at an excitation of 488 nm
K = Koko
B = Bernhard
P = Promega S30 for linear templates
Lysate | Premix | Amino Acids | Feed? | comment |
---|---|---|---|---|
K | K | ours | Mg(OAc)2 | - |
K | K | ours | no | - |
B | K | ours | Mg(OAc)2 | - |
B | K | ours | no | - |
B | P | P | no | - |
P | P | P | no | positive control |
K | H2O | H2O | no | negative control |
- short: BK = lysate Bernhard, Premix Koko
- the negative control contains KK and no DNA
- repetition of gel control of pIG15_104 and 105 by loading 20 µl on a gel (Luisa)
- Purification of PCR product of aminylated linear 104 and 105 (Sabi)
- elution with 5 µl water
- Purfication of PCR product of linear 104 and 105
- Elution with 12,5 µl water
- Miniprep of pIG15_104, pIG15_105 and PQE HA-GFP-His6-His6
- Elution with 25µl
== PCR for linear template of pIG15_1104 (His10-C.tetani-Spy) ==
(Luisa)
* length 1371 bp
* primers oIG15_s001 and pIG15_s003
* program
^ Temperature °C ^ duration ^
|98|5 min |
|98|20 sec |
|52 °C |20 sec |
|72 |70 sec |
|72 |5 |
|4 |infinite |
* 1 reaction, 50 µl
^ Component ^ amount [µl] ^
|buffer - Phusion HF|10 |
|dNTPs| 2.5 |
|Primer forward |4.4 |
|Primer reverse |4.4 |
|Phusion Polymerase |0.5 |
|Template (pre-purified) <250 ng |1 |
|DMSO | 1.25 |
|H2O to 100µl each | 26.75 |
(not yet performed)
Thu, 06.08.2015
PCR for high yield template of pIG15_105 and pIG15_a105 (Amino-His10-tYFP-Halo)
- was performed with 50 µl aliquots of a 500 µl master mix using 1.25 µl DMSO and 2.5 µl dNTPs
- for aminylated PCR product 1 x 50 µl reaction was tested
- increased elongation time to 120 sec
- program pIG15_105
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
52 °C | 20 sec |
72 | 120 sec |
72 | 5 |
4 | infinite |
- Master Mix
Component | amount [µl] |
---|---|
buffer - Phusion HF | 110 |
dNTPs | 27.5 |
Primer forward | 44 |
Primer reverse | 44 |
Phusion Polymerase | 0.5 per tube |
Template (pre-purified) <250 ng | 11 |
DMSO | 13.75 |
H2O to 100µl each | 294.25 |
- for pIG15_a105
Component | amount [µl] |
---|---|
buffer - Phusion HF | 10 |
dNTPs | 2.5 |
Primer forward | 4 |
Primer reverse | 4 |
Phusion Polymerase | 0.5 |
Template (pre-purified) <250 ng | 1 |
DMSO | 1.25 |
H2O to 100µl each | 26.75 |
PCR for linear template of pIG15_104 (His10-tYFP-Spy), pIG15_a104 (Amino-His10-tYFP-Spy)
- for regular pIG15_104 the PCR was performed in 10 x 50 µl reactions with an increased elongation time
- for aminylated PCR product 1 x 50 µl reaction was tested
- program pIG15_104 and pIG15_a104
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
52 °C | 20 sec |
72 | 60 sec |
72 | 5 |
4 | infinite |
→ elongation time was increased to 60 seconds
- Master Mix pIG15_104
Component | amount [µl] |
---|---|
buffer - Phusion HF | 110 |
dNTPs | 27.5 |
Primer forward | 44 |
Primer reverse | 44 |
Phusion Polymerase | 1 per tube |
Template (pre-purified) <250 ng | 11 |
DMSO | 13.75 |
H2O to 100µl each | 294.25 |
- for pIG15_a104
Component | amount [µl] |
---|---|
buffer - Phusion HF | 10 |
dNTPs | 2.5 |
Primer forward | 4 |
Primer reverse | 4 |
Phusion Polymerase | 0.5 |
Template (pre-purified) <250 ng | 1 |
DMSO | 1.25 |
H2O to 100µl each | 26.75 |
Luciferase Dilution Reagent
- adapted from Promega S30 Kit for linear templates
concentration | component | comment |
---|---|---|
25 mM | Tris-Phosphate (pH 7.8) | |
2 mM | DTT | |
2 mM | edta | in original protocol: 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid |
10 % | glycerol | |
1 % | Triton X-100 | |
1 mg/ml | BSA |
Wed, 05.08.2015
western blot and dot blot started yesterday 4.8.15:
- 3x washing with TBS-T
- 1nd Antibody:
- anti GFP Mouse 1:3000 in TBS-T
- anti-Luc mouse 1:500 in TBS-T
- 3x washing with TBS-T
- 2nd AB: anti Mouse 1:20000
- 3X washing with TBS-T
- next time gel should be run at increased Ampère. In the applied running time, the bands didn't separate well
- perhaps a change of gel concentration would be beneficial as well
Ponceau staining of western membranes
- Dot blot shows some clear protein spots
- not a lot of protein is visible in the lines of the western blot
- some of the samples weren't visible at all –> repeat of western necessary → preparation of bigger samples necessary
Western blot(transmitted light picture; EcL-Picture[3 min]; picture of arrangement taken with camera
)
- Anti-GFP shows bands in the lane of B+P –> however bands are at 15kDa it should be at 27kDa
Dot Blot-GFP (transmitted light picture; EcL-Picture[3 min]; picture of arrangement taken with camera)
Dot blot LUC (transmitted light picture; EcL-Picture[3 min]; picture of arrangement taken with camera)
PCR for linear template of HA-GFP-His6-His6
(Luisa)
- test HA-GFP-His6-His6 PCR in 6 x 25 µl reactions
Sample | buffer | DMSO [µl] | H2O [µl] |
---|---|---|---|
1 | Phusion HF | 0 | 12.875 |
2 | Phusion HF | 0.5 | 13.375 |
3 | Phusion HF | 1 | 13.875 |
4 | Phusion GC | 0 | 12.875 |
5 | Phusion GC | 0.5 | 13.375 |
6 | Phusion GC | 1 | 13.875 |
- program HA-GFP-His6-His6
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
69 | 20 sec |
72 | 60 sec |
72 | 5 |
4 | infinite |
- Master Mix
Component | amount [µl] |
---|---|
buffer - Phusion HF or GC | 15.1 each |
dNTPs | - |
Primer forward | 6.04 each |
Primer reverse | 6.04 each |
Phusion Polymerase | 0.755 each |
Template (pre-purified) <250 ng | 1.51 each |
DMSO | - |
H2O to 100µl each | - |
- elongation time was increased to 1 minute
- 10 µl of PCR product were tested on a gel
- from now on HA-GFP-His6-His6 PCRs should be performed using no DMSO and Phusion GC buffer
PCR for high yield template of HA-GFP-His6-His6
(Luisa)
- the PCR will be performed in 10 x 50 µl reactions, while loading 10 µl on a gel as control
- program HA-GFP-His6-His6
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
69 | 20 sec |
72 | 60 sec |
72 | 5 |
4 | infinite |
- Master Mix
Component | amount [µl] |
---|---|
buffer - Phusion GC | 110 |
dNTPs | 27.5 |
Primer forward | 44 |
Primer reverse | 44 |
Phusion Polymerase | 5.5 |
Template (pre-purified) <250 ng | 11 |
DMSO | - |
H2O to 100µl each | 294.25 |
Tue, 04.08.2015
cellfree expression of digested GFP and luciferase
(both)
- compare expression of GFP and Luciferase in:
- Bernhard lysate + own amino acids + own premix + single components (PEP, ATP, GTP, CTP, UTP, tRNA, CoA) + 2000 ng GFP and 1500 ng luciferase DNA in DECP water
- Bernhard lysate + promega amino acids + promega premix + 2000 ng GFP and 1500 ng luciferase DNA in DECP water
- promega lysate + promega amino acids + promega premix + 2000 ng GFP and 1500 ng luciferase DNA in DECP water
- negative controls:
- own lysate following EMBL protocol (Koko) + own amino acids + own premix + DECP water, no DNA
- promega lysate + promega amino acids + promega premix + DECP water, no DNA
- reaction was performed at 30 °C, 300 rpm for 2h in the thermomixer
- afterwards samples were kept at 4°C
PCR for linear template of pIG15_105 and HA-GFP-His6-His6
(Luisa)
- test HA-GFP-His6-His6 PCR from yesterday by loading 20 µl to a gel
- gel control of pIG15_105 PCR from yesterday
- purification of pIG15_105 PCR (Sabi):
- elution in: 12,5 ul on 2 columns
- concentration: 950 ng/µl
- Western Blot of expression of pIG15_105 and HA-GFP-His6-His6 from today:
- big 12 % SDS Gel with Collecting gel was cast ( next time use of red comb!)
- 24 ul of the GFP samples and negative controls were mixed with 6ul SDS 5X running buffer and heated to 95 °C for 10 min
- for Luc samples: due to not enough sample after back and forth pipetting on the plate only 20 µl sample were used
- 15 ul of the samples were put onto the blot. However pipetting was quite difficult due to solid components in the sample and inhomogenity ( perhaps next time the use of more sample is necessary. Also shering of samples with thin pipette could be tried)
- gel run for ca 2 h at 0,02 A
- Blotting of Gel for 1,5 h at 0,12 A
- Gel was stained with Comassie oN
- blot was blocked oN at 4°C( together with the dot blots) in 5% milk powder TBS-T
- Dot Blot of expression of pIG15_105 and HA-GFP-His6-His6 from today (Sabi):
- pipetting scheme : marked with a pen on the blot
- 1µl of the samples were pipetted onto the methanol-activated PVDF membrane
- Membrane was still slightly wet or rewetted for the drops to be able to seep in
- with SDS buffer prepared samples were spotted onto the membrane
- additionally non treated samples were spotted onto the membrane
- for Luciferase: samples that were diluted and mixed with reaction reagent from the plate reader were used
- Membrane was blocked with TBS-T with 5% milk powder oN
Mon, 03.08.2015
- Dot blot of Expression of pIG15_105 and HA-GFP-His6-His6 from yesterday: (Sabi)
- pipetting scheme
- 2ul of the samples were pipetted onto the methanol-activated PVDF membrane
- Membrane still has to be slightly wet or drops aren`t able to seep into the membrane
- Membrane was blocked with TBS-T with 5% milk powder for 1 h (and specific blocking buffer for His-Conjugate)
- 3 X washing with TBS-T
- First AB for 2 h in TBS-T:
- anti-tYFP 1:5000 in TBS-T Rabbit polyclonal
- anti LUC 1:500 in TBS-T Mouse monoclonal
- anti His-Conjugate (Qiagen) incubated on membrane until the other blots were ready for final washing steps
- 2nd AB for 1 h in TBS-T:
- anti_Mouse (1:20000) for Luc
- anti- Rabbit (1:20000) for tYFP
- 3 X washing with TBS-T
- Blots in Reader: YFP ( left up); Luc (right up); His (left down)
PCRs of pIG15_105 and HA-GFP-His6-His6
(Luisa)
- PCRs will be performed in 6 x 50 µl reactions with varying dNTP and DMSO concentrations
Sample | dNTPs [µl] | DMSO [µl] |
---|---|---|
1 | 2.5 | 1.25 |
2 | 2.5 | 1.5 |
3 | 2.5 | 1.75 |
4 | 3 | 1.25 |
5 | 4 | 1.25 |
6 | 5 | 1.25 |
- program HA-GFP-His6-His6
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
69 | 20 sec |
72 | 30 sec |
72 | 5 |
4 | infinite |
- program pIG15_105
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
52 °C | 20 sec |
72 | 70 sec |
72 | 5 |
4 | infinite |
- Master Mix
Component | amount [µl] |
---|---|
buffer - Phusion HF | 66 each |
dNTPs | - |
Primer forward | 26.4 each |
Primer reverse | 26.4 each |
Phusion Polymerase | 0.5 per tube |
Template (pre-purified) <250 ng | 6.6 each |
DMSO | - |
H2O to 100µl each | 176.55 each |
- PCR for HA-GFP-His6-His6 did not work because of problems with the thermocycler
- PCR was repeated with protocol above, with addition of a 7th sample containing 1.25 µl DMSO, 2.5 µl dNTPs and Phusion GC buffer instead of Phusion HF buffer
- final PCR for pIG15_105 was performed with 50 µl aliquots of a 500 µl master mix using 1.25 µl DMSO and 2.5 µl dNTPs.
- program pIG15_105
Temperature °C | duration |
---|---|
98 | 5 min |
98 | 20 sec |
52 °C | 20 sec |
72 | 70 sec |
72 | 5 |
4 | over night |
- Master Mix
Component | amount [µl] |
---|---|
buffer - Phusion HF | 110 |
dNTPs | 27.5 |
Primer forward | 44 |
Primer reverse | 44 |
Phusion Polymerase | 0.5 per tube |
Template (pre-purified) <250 ng | 11 |
DMSO | 13.75 |
H2O to 100µl each | 294.25 |
Sun, 02.08.2015
(Sabi)
- Purification and Concentration of lin PQE-HA-GFP-2(HIS)6 with the Qiagen Kit
- 0,5 ml were purified on two columns and eluted with a 10ul
- only 100ng/ul
- improvement of PCR protocol necessary as the controlgel also shows some degradation
- Cellfree Expression of lin PIG15_104 and Luciferse
- Reactions were performed in tubes
- 50 ul reactions at 30°C for 2h
- part of the reactions were feeded every 20 min with a 0,5 ul Mg solution
- after completion of reaction samples were transfered to a white 96 well plate for measurement of luminescence and fluorescence measurement
- the single parts of the reaction mix were useed in relation to the promega mix
Amino Acids | 6,00 |
---|---|
S30 Premix | 20,00 |
S30 Extrakt | 15,00 |
Template | x (1300ng) |
H20 | a.D. 50 ul |
- In several of the reactions parts of the kit were exchange for our own mixes to test the quality of the single components
Pipetting sheme
8 | 9 | 10 | 11 | 12 | |
---|---|---|---|---|---|
A | Promega Kit (luc 1:2) | Promega Kit with our Premix (Luc 1:2) | Promega Kit with our Aminoacids (Luc 1:2) | Promega Kit with our Berni Lyaste(Luc 1:2) | Bernhard reaction (Luc 1:2) |
B | |||||
C | Promega lin 104 | Promega lin 104 +feed | Bernhard lin 104 | Bernhard lin 104 +feed | |
D | Promega with Berni Lysat lin 104 | Promega with Berni Lysat lin 104 +feed lin 104 | Promega neg. |
* Analysis:
- feeding didn`t change the outcome
- expression of luciferase probably didn`t work–> signal usually at 580nm
- no expression anywhere –> problems with DNA
- repetition of experiment necessary
Sat, 01.08.2015
(Sabi)
- Purification and concentration of linIG15_104 with Qiagen Kit
- 104_: 750ng/ul
- pOE-Ha-GFP-2xHis: 60ng/l
- repeat of PCR from tuesday with an 1:10 Dilution of the purified product