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)

• 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)

(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

(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

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:

(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)

(Luisa)

• 5 columns per sample, elution in 12 µl each, pooled for measurement

(Luisa)

(Luisa)

blots are so bad they have to be repeated anyways. therefore i did not waste any time making the pictures pretty..

(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

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

(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

(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

• 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.

• 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:

• elution in 22 µl nuclease free water
• concentration: 389 ng/µl

• 2 x 250 ml were incubated over night

• 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

• 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^

 
  * Gel2: 

* 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

• 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

• tube reactions are measured in plate reader at 488nm/500nm
• a western blot with GFP antibody will be performed

• gel loading pattern: (12% sds-page, 0,? A, 1:30h)

• 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?

(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

(Luisa)

• 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

(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

(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

→ 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

• TO DO (Koko)

WesternBlot of the //HA-GFP-His6-His6// from Wed, 12.08

make moar plasmid! (pQEHA-GFP-His6-His6)

collaboration with SurRIf subgroup.

• spotting onto Ni-NTA surfaces, by hand, using a stencil for uniformity of the spots (made of PDMS)

spotting pattern:

• 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:

Results:

Roi selection Exp. 46b: Spot 11 couldnt be selected due to air bubble on it

Binding curves of Exp. 46b for all spots

Binding curves of Exp. 46b only for spots 1-6

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….

(Luisa)

• 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

• 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

• 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

• Results

(Koko)

• 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

• 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

(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

• 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)

• 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

• 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

• 5 expressions and a negative control in 50µl size were carried out in parallel:
  • Mastermix for 5 rx:

• 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)

• 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

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

• 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)

• 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

• Master Mix

• for pIG15_a105

• 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

→ elongation time was increased to 60 seconds

• Master Mix pIG15_104

• for pIG15_a104

• adapted from Promega S30 Kit for linear templates

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

Comassie of Western Blot

• 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)

(Luisa)

• test HA-GFP-His6-His6 PCR in 6 x 25 µl reactions

• program HA-GFP-His6-His6

• Master Mix

• 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

(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

• Master Mix

(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

(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

• 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)
  • 

(Luisa)

• PCRs will be performed in 6 x 50 µl reactions with varying dNTP and DMSO concentrations

• program HA-GFP-His6-His6

• program pIG15_105

• Master Mix

• 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

• Master Mix

(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

• 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

* 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

(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