Team:Freiburg/Labjournals/Cellfree/June
Labjournal Cellfree June
Tue, 30.06.2015
- Mini prep of pJet with Halo PCR-Product was repeated
- buffer two of biozym miniprep kit ran out and buffer two of roche kit was substituted
- miniprep did not work, product not pure, poor 260/280 values and no DNA-resembling curve on NanoDrop
Mon, 29.06.2015
- Mini Pep of pJet with Halo PCR-Product
- 4 Different Clones were prepped
- unfortunately ethnol-containing buffer made inscriptions invisible –> numbers don't match plates any more
- each four clones were picked from the classical cloning and cloning plate. For further identification numbers were added.
Sun, 28.06.2015
- Cloning of pIG15_105 approaches:
- pJET with Halo-Tag: Picking of Clones from Trafo
- Fusion PCR approach –> no correct bands after test digest –> failed
- Gibson: small colonies –> transfer tomorrow
- classical cloning: only 30 minute ligation at night, so one part was transformed, while the other part proceeded with ligation @4°C
Sat, 27.06.2015
- Continuation of Western blot from 26.06.2015
- 1st antibody incubation. 1h @ RT (AB-dilution 1:1 ,000 in blocking buffer with 1% milk powder)
- only Anti-Gfp (monoclonal) was used ( AB received from Phillip Schwenk)
- Washing membrane 3x 15 min in blotting buffer
- 2nd antibody incubation. 1 h @ RT (AB-dilution 1: 10,000 in Blotting buffer anti-mouse, 1:20,000for ECL)
- Washing membrane 3x 10min in blotting buffer
- mesuring in ECL reader for 7,5 min (every 30 sec)in serial mode –> only noise on picture
- measuring for 3 min with 8×8 binning –> empty blot
- new approaches for pIG15_104:
- blunt end ligation of halo and pJET for better chances in restriction digestion → repetition of halo pcr
- fusion pcr of pIG15_104 and halo from original plasmid
- Gibson of pIG15_104 and halo pcr product
- classical: digestion of halo pcr product and pIG15_104
- 30 minute ligation,transformation _> rest left in fridge over night at 4°C for second transformation
- ligation of empty vector as control
Fri, 26.06.2015
- Westernblot of first and second cellfree Expression
- 20 µl per sample (16µl probe and 4µl Sds-Puffer) besides neg control of first expression there were only 10µl left
- pipetting sheme
| YFP | GFP | negativ | marker | YFP | GFP | negativ |
|---|---|---|---|---|---|---|
| 1.EX | 1.Ex | 1.Ex | - | 2.Ex | 2.Ex | 2.Ex |
- Cutting of PVDF membrane and 4 whatman-papers in gel size
- PVDF membranes were activated in 100% MeOH before use
- Semidry blotting
- Stack: 2x Whatmann, Filter, Gel, 2x Whatman
- everything was soaked in blotting buffer prior to use
- (no bubbles after adding each layer)
- reduction of buffer necessary due to electrical short
- Blot for at 0,2 mA <note tip>tip</note>h (0.8mA/cm2 )
- Blot was dismanteled
- whatmann paper were washed and drieed for reuse
- Gel was stained with Comassie and dried in Ethanol solution for storage
- Block membrane ca 30 min in blocking buffer (Blottin Buffer with 5% Milk pulver)
- Washing with Blotting Buffer 3 x 15 min
- last wash overnight @ 4°C
Thu, 25.06.2015
- Minipreps of the 8 colonies
- Control digest of possible pIG15_105 and gel with SpeI-HF and HindIII-HF (expected band size:1077 bp 2935 bp)
- 2nd cellfree expression
- 100µl per reaction, 3´.5 h duration @37°C in the thermomixer with 300rpm
- as DNA templates:
- pQE-60 with GFP-His from the toolbox as outside standard (164ng/µl)
- pIG15_104 with HIS-tYfp-Spy (134ng/µl)
- and water as negative control
- everything was pipetted on ice reaction was stared by addition of DNA and immiditae placement in the thermomixer
| DNA | 1x [µl] | 3x [µl] |
|---|---|---|
| Premix | 10 | 30 |
| Amino Acids | 10 | 30 |
| PEP | 4,2 | 12,6 |
| CoA | 1,212 | 3,636 |
| tRNA | 17,5 | 52,5 |
| ATP | 2,5 | 7,5 |
| CTP | 1,7 | 5,1 |
| GTP | 1,7 | 5,1 |
| UTP | 1,7 | 5,1 |
| Lysate | 24 | 72 |
| DNA | DNA [µl] | Water [µl] | Mastermix |
|---|---|---|---|
| pIG15_104 | 11,194 | 14,294 | 74,512 |
| pQE-60 | 9,146 | 16,342 | 74,512 |
| -Con | 0 | 25,488 | 74,512 |
- Afterwards 0,5 µl were pipetted on a objectslide and analyzed under the fluoresence microscope (Gfp filter)
- only GFP and negative control were observed
- clear difference to negative control
- pictures of GFP –> saved as nef format (in future Tiff)
- Problems with to ast bleaching for taking a photo
- variation of shutter speed etc.
- Rest of the samples was frozen at -20°C
Wed, 24.06.2015
- 1st cellfree expression
- 25 µl per reaction, 2 h duration @37°C in the thermomixer with 300rpm
- As DNA templates:
- pQE-60 with GFP-His from the toolbox as outside standard (164ng/µl)
- pIG15_104 with HIS-tYfp-Spy (134ng/µl)
- and water as negative control
- everything was pipetted on ice reaction was stared by addition of DNA and immiditate placement in the thermomixer
| DNA | 1x [µl] | 3x [µl] |
|---|---|---|
| Premix | 2,5 | 7,5 |
| Amino Acids | 2,5 | 7,5 |
| PEP | 1,05 | 3,15 |
| CoA | 0,303 | 0,909 |
| tRNA | 4,375 | 13,125 |
| ATP | 0,625 | 1,875 |
| CTP | 0,425 | 1,27 |
| GTP | 0,425 | 1,275 |
| UTP | 0,425 | 1,275 |
| Lysate | 6 | 18 |
| DNA | DNA [µl] | Water [µl] | Mastermix |
|---|---|---|---|
| pIG15_104 | 3,760 | 3,760 | 18,628 |
| pQE-60 | 2,134 | 4,238 | 18,628 |
| neg control | 0 | 6,372 | 18,628 |
- Afterwards 0,5 µl were pipetted on coverslip and analyzed under the fluoresence microscope (Gfp filter)
- only GFP and negative control were observed
- both shine green without differnence
- Rest of the samples was frozen at -80°C
Tue, 23.06.2015
- Picking of 8 colonied from all plates with the different cloning strategies
Mon, 22.06.2015
- Trafo of overnight ligation for pIG15_105
Digest of pJET with Halo-Tag insert
- two step process with first one hour with AflII and than one hour HindIII
- more DNA used for sigest (1µg Halo and 0,5µg Backbone)
- digest of pIG15_104 in two steps
- Ligation of new digest
- Trafo of new digest
Sun, 21.06.2015
- over day digest of Halo in pJET1.2 blunt vector for pIG15_105
Fri, 19.06.2015
- sending clones out for sequencing
- preparing extract buffers with DTT and ßME
- ligation of HaloTag into pJET1.2 blunt vector from yesterday * failed due to wrong buffer or no cells in transformation mix
- ligation of HaloTag into pJET1.2 blunt vector
Thu, 18.06.2015
sending clones out for sequencing:
- preparing extract buffers with DTT and ßME
- ligation of HaloTag into pJET1.2 blunt vector
Wed, 17.06.2015
lab today slightly less occupied by strange forces from other worlds.
- miniprep of all 4 A2 + halo (pIG15_105)</todo>
- comment:
- 1: 67,2 mg/ml
- 2: 78,8 mg/ml
- 3: 66,1 mg/ml
- 4: 60,1 mg/ml
- control digest of pIG15_105 with HindIII and Afl II
- control with gel:
at all digests: visible band at 3kb, which would be the size of the digested backbone, but no insert (a.k.a. HaloTag) at expected 880bp visible on the gel.
- today the final chemicals arrived: folinic acid, cAMP, K-glutamate and the 20-L-Amino-Acid kit from Sigma were given to us from thr AG Roth.
- the aminos were then dissolved to 80 mM, which is 10x the concentration of the stock of the „Bernhard-protocol“ and stored at 4°C in a 15ml falcon.
- 1l of the culture needed for the cellfree extract-preparation was prepared following the EMBL protocol.
Tue, 16.06.2015
our lab was still occupied by strange forces from other worlds.
- replating of four colonies on plate 1 of transformation (A2 + halo)
- miniprep of A2 liquid colonies
Mon, 15.06.2015
today our lab was occupied by strange forces from other worlds.
- both HaloTag PCR-clean-up and the original A2 plasmid backbone were digested
| HaloTag insert | |
|---|---|
| component | amount [µl] |
| plasmid (concentration=47,8ng/µl) | 2 |
| CutSmart buffer | 2 |
| Hind III HF | 1 |
| Afl II | 1 |
| ddH2O ad 10µl | 4 |
| A2 backbone | |
|---|---|
| component | amount [µl] |
| plasmid (concentration=45ng/µl) | 2 |
| CutSmart buffer | 2 |
| Hind III HF | 1 |
| Afl II | 1 |
| Phosphatase Buffer | 2 |
| Antarctic Phosphatase | 1 |
| ddH2O to 20µl | 1 |
- The digested parts were then ligated:
| component | amount [µl] |
|---|---|
| plasmid backbone | 2 |
| plasmid insert | 16 |
| T4 DNA Ligase buffer | 4 (because old) |
| T4 DNA Ligase | 1 |
Ligation performed at RT for 1h.
- The A2 backbone alone was religated as negative control.
| component | amount [µl] |
|---|---|
| plasmid backbone | 1 |
| T4 DNA Ligase buffer | 2 |
| T4 DNA Ligase | 1 |
| H2O ad 20 | 17 |
- A transformation was performed using 25µl TOP10 competent cells with 10µl of plasmid-ligation product following our standard protocol. 2 plates were streaked and are grown o/n.
- To produce more of the plasmid backbone, a liquid culture of the A2 clone was inoculated.
Sun, 14.06.2015
- gelelectrophoresis of pcr product showed no/ wrong bands.
lane1: empty - lane2: 2log dna ladder - lane3+4: sample+ midoridirect - lane5: ladder - lane6:empty
- repeating PCR, using same amounts and program as yesterday Sat, 13.06.
- repeating gelelectrophoresis (1% agarose), this time using in-gel staining via 4µl MidoriGreen Adavnce instead of sample-loaded staining.
- second PCR and gelectrophoresis showed the right band at about 880 bp:
- band was cut out and purified using qiaquick gelextraction kit. (30µl water instead of elution buffer)
- only 5µl of the PCR product was loaded onto the gel, the other 15µl were directly purified using the Zymo DNA-Clean&Concentration Kit.
- Changes: Instead of 6µl elution buffer, 8µl of warm water was used. After that, the column was eluted for a second time with the same amount of water. The second elution still contained 10ng/µl of DNA.
Sat, 13.06.2015
Due to lack of growth on plates, restart of procedure from PCR of HaloTag onwards.
- Template dilution 1:20 → 1,4 ng/µl
* PCR of Halo tag
| Temperature °C | duration |
|---|---|
| 98 | 5 min |
| 98 | 10 sec |
| 63.5 | 20 sec |
| 72 | 22 sec |
| 72 | 5 |
| 4 | infinite |
| buffer - Phusion HF | 5 |
| dNTPs | 0,5 |
| Primer forward | 1,25 |
| Primer reverse | 1,25 |
| Phusion Polymerase | 0,25 |
| Template <250 ng | 1 |
| H2O to 25µl | 10,75 |
Primers used: 105f + 106r
Product placed in cooler at 4°C.
Thu, 11.06.2015
no growth on plates. Transformation repeated with 25µl aliquot of TOP10 cells.
Wed, 10.06.2015
- sequencing results for sample two of both plates were positive
- both clones 2 (plate A and B) were replated onto new plates
- Test Digestion of Halo PCR cleanup product and „A2“ clone plasmid
with Afl I + Hind III, following standard amounts
- Separation using 1% Agarose Gel, 120V, 40min
- Imaging showed two clear bands, each one at the right size: the halo product around 800bp, the linearized backbone of the A2 clone backbone at 3000bp:
- Gelextraction using Qiagen Qiaquick Kit, elution with 30µl ddH20 at 30°C
concentration: 6,6ng/µl for Halo digest, 45ng/µl for A2 digest
- Ligation: 30µl of Halo plasmid, 1µl of A2 plasmid, 3h at RT
- Transformation into 100µl of TOP10 competent cells
Tue, 02.06.2015
- pIG_003 sequencing didn't work bc primers with gibson overhang were mistakingly chosen
- pIG_003 sequencing is repeated with cloning group primers oIG_s001 ans oIG_s003
Tue, 02.06.2015
- Halo tag pcr didn't work → wrong bands on gel
- Template dilution 1:20 → 1,4 ng/µl
* PCR of Halo tag
| Temperature °C | duration |
|---|---|
| 98 | 5 min |
| 98 | 10 sec |
| 63.5 | 20 sec |
| 72 | 22 sec |
| 72 | 5 |
| 4 | infinite |
| buffer - Phusion HF | 5 |
| dNTPs | 0,5 |
| Primer forward | 1,25 |
| Primer reverse | 1,25 |
| Phusion Polymerase | 0,25 |
| Template <250 ng | 1 |
| H2O to 25µl | 10,75 |
- PCR product was loaded on 1% agarose gel and let run for 35min at 120V
though no ladder was visible, the only visible band was cut out from the gel and extracted using the QiaQuick Gel-Extraction Kit.
- The elution should now contain the Halo tag
- pIG_003 was sent out for sequencing
Mon, 01.06.2015:
- miniprep of colonies from g-block + pSB1C3 ligation
- control digest, gel
- prepped plasmids from the colonies were prepared for sequencing
- PCR of Halo tag
| Temperature °C | duration |
|---|---|
| 98 | 5 min |
| 98 | 10 sec |
| 63 | 20 sec |
| 72 | 40 sec |
| 72 | 5 |
| 4 | infinite |
Sun, 31.05.2015:
* Re-ligation of pIG15_001 backbone with gBlock inser * Transformation of overnight-ligation from Fri, 29.05 in TOP10 cells
- checking for colonies: both plates grew about 20 - 50 colonies. 4 colonies were picked from each plate and re-plated.
