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Amplifying of biobricks by PCR
Start by mixing the receipt of PCR from beneath, multiply by the amount of PCR reactions. Add the DNA template to the PCR tubes and add with PCR mix.
PCR MIX
| PCR mix |
1 x PCR mix á 50µl |
| 5 x HF PCR buffer with MgCl2 or GC buffer |
10µl |
| dNTP’s 2mM |
5µl |
| Primer forward 10 µM |
4µl |
| Primer reverse 10 µM |
4µl |
| Phusion DNA polymerase 5u/µl |
0.3µl |
| DNA template |
0.5µl |
| MilliQ water |
26.20µl |
PCR Programs
| Temperature [°C] |
Time [min] |
Cycles |
| 98 |
2:00 |
|
| 98 |
0:10 |
35 |
| 59 |
0:30 |
- |
| 72 |
3:00 |
- |
| 72 |
5:00 |
|
| 12 |
Store |
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To check the PCR reactions were correct, all PCR samples were run on a Gel electrophoresis. All PCR reactions of 50µl were added 7-10µl loading buffer and run in 2% agarose. The Gel electrophoresis was set the volt to 75V and time after length but between 30-60 minutes.
Purification of PCR Product
Gel electrophoresis
Place 20 µl of --- in 4-5 drops on the casting tray. Add agarose gel and mix until even. Let the gel cure for 30-45 min.
On a piece of parafilm, mix 1 µl of PCR product with 3 µl of loading buffer.
Load the gel with the samples, a negative, and DNA ladder.
Run the gel at 70V. The time is depending on the bp length, but 30-60 min are usually good for 1000-3000bp.
Visualise the gel in a ----.
USER cloning
Start mixing the USER mix, from the table beneath. Muliplying by the number of USER-clonings that have to be run. PCR product are not put in yet.
USER mix
| USER mix |
1 x USER mix á 10µl |
| USER enzyme |
1 µl |
| NEB (10 x diluted) |
0,5 µl |
| BSA |
0,5 µl |
| PCR product |
8 µl |
Transfer 2 µl of the USER mix to PCR tubes.
Add the PCD products in an amount equal to 8µl for all components.
Incubate for 37°C for 40 min and 30 min at 25°C.
Tranformation in E.coli
The preparations for the transformation can preferably be done while the USER cloning is incubating.
Take LB- plates out of the refrigerator and mark them. Remember to use LB-plates with the right antibiotic.
Take 50 µl competent "E. coli DHα5" cells per USER reaction, from the -80 freezer and place on ice. Additionally, place 1,5 ml tubes on ice.
Add all USER reaction to the 50µl tube with the competent "E. coli" cells. Mix well by pipetting.
Keep cells on ice for 30 min.
Turn on the hot plate at 60°C.
Heat chock each transformation for 90 sec. Afterward, put directly on ice for 2 min
Plating of bacteria on LB-plates.
Bacteria backbone with amp resistence gene.
Sterilize a Drigalski spartula in 90% ethanol and flame between each transformation. Plate all the transformation mix out on the LB-amp plate and disperse with the cooled drigalski.
Bacteria backbone without amp resistence gene
-Add 500 µl LB to the transformation mix.
-Incubate for 30-60 min at RT.
-Spin the transformation mix down.
-Remove the supernantant until approximately 50 µl are left.
-Resuspend the pellet in the remaining LB.
-Plate the 50 µl of tranformatin mix in the same way as described above, but on LB plate with the specific resistance gene.
Incubate over night at 37°C.
Next day; Check for visual colonies and use them for cultivating.
Cultivation of transformed cells
Choose a couple of colonies from the LB-plate.
Each colony are transferred to 5 ml LB + resistance marker with a pipet tip.
Incubate over night at 37°C in the shaking incubator.
Purification of plasmids
The GenElude Plasmid Miniprep Kit from Sigma-Aldrich was used to isolate our plasmids to use in fungi while QIAGENs EndoFree Plasmid Maxi kit was used to purify plasmids for use in mammalian cells.
Sigma-Aldrich protocol (use in fungi).
Harvest cells:
Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E. coli culture to a microcentrifuge tube and pellet cells at ;12,000 3 g for 1 minute. Discard the supernatant.
Resuspend cells:
Completely resuspend the bacterial pellet with 200 μl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous. Incomplete resuspension will result in poor recovery.
Lyse cells:
Lyse the resuspended cells by adding 200 μl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous. The lyse reaction must not exceed 5 min.
Neutralize:
Precipitate the cell debris by adding 350 μl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifuging at ≥12,000*3 g or maximum speed for 10 minutes. Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out of solution as a cloudy, viscous precipitate.
Prepare columns:
Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 μl of the Column Preparation Solution to each miniprep column and centrifuge at ≥12,000*3 g for 30 seconds to 1 minute. Discard the flow-through liquid.
Load cleared lysate:
Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at ≥12,000*3 g for 30 seconds to 1 minute. Discard the flow-through liquid.
Wash column:
Add 750 μl of the diluted Wash Solution to the column. Centrifuge at ≥12,000*3 g for 30 seconds to 1 minute. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol.
Elute DNA:
Transfer the column to a fresh collection tube. Add 100 μl of MilliQ water to the column. Centrifuge at ≥12,000*3 g for 1 minute.
The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C.
Fungi
Transformation in fungi
Genetic Transformation of filamentous fungi – protocol from Center for Microbial Biotechnology (CMB) at DTU, Author Nielsen, J. B.
Media:
Minimal medium (MM) (1L) -50 mL D-glucose 20% w/V, 20 mL 50x mineral mix, 10 mL 1 M sodium nitrate, 20 g ager.
Transformation media(TM)(1L)- 342.3 g Sucrose, 20 mL 50x mineral mix, 20 g agar
Mineral Mix (1L)- 26g KCL, 26g MgSO4·7H2O, 76g KH2PO4, 50 mL Trace element solution, MIlli-Q water to volume 1000 mL
D-glucose 20% (0.5 L) - 100g D-glucose and MilliQ water up to 500 ml
Aspergillus protoplastationbuffer (APB)- Final conc. 1.1 M MgSO4 and 10 mM Na-phosphate buffer. pH is adjusted with 2 N NaOH to 5.8.
Aspergillus transformation buffer (ATB)- Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.
PCT (200ml) - Final conc: 50% w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store at 4 °C
Initiation: The host strain is grown as three-point stabs on Minimal medium plates with the require suppliants added. MM will for convenience throughout the protocol refer to MM with the supplements included.
Inoculation: The conidia are harvest by adding 5 ml of MM and firmly rub with a sterile Drigalsky spatula. The conidial suspension is pipette to a sterile 500 ml shake flask containing 100 ml MM. The cultures are incubated at 30 °C with 150 rpm of shaking over night (14-20 hours)
Mycelial harvest: A funnel with a sterile Mira cloth (filter) is used to harvest mycelia. To remove residual glucose from mycelia the biomass are wash with Aspergillus protoplastationbuffer (APB). The filtered biomass is transferred to a new Falcon tube with a sterile spoon.
Protoplastation: Mycellium is resuspenden in 10 ml filter-sterillized(0.45μm filters) APB containing 40 mg Glucanex/ml. The Glucanex is dissolved in APB with gentle magnetic stirring less than 100/min. Mycelia with dissolved Glucanex are mixed at 30 °C with 150 rpm of shaking for 2-3 hours.
Portoplast solutions are diluted in APB adding up to 40 ml mark. An overlay of max. 5ml Aspergillus transformation buffer (ATB) diluted to ½x with sterile MilliQ-water is carefully placed on top of the APB. Centrifuged at 13 min 3000 RCF in Sorvall centrifuge. Protoplates should be observed as a halo of with slurry in the interphase of the two liquids. Withdraw of the protoplasts are done with pipette and placed in a Falcon tube. ATB is added up to 40 ml mark. Centrifuge at 300 RCF in 13 min and supernatanted are discarded.
The protoplastes are resuspended in 1 ml ATB with a 5 ml pipette.
Genetic transformation: Aliquotes of 50 μl are transferred to a 1.5 ml Eppendorf tube containgen 10 μl of DNA for transformation. Protoplast and DNA are incubated at room temperature for at least 30 min.
Protoplat and DNA suspension are added to 1 ml PCT in a 15 ml tube and shake gently. Incubated for 1-5 min at room temperature. Diluted in 3 ml ATB. The tube is filled with molten transformation medium (TM) agar (temperature of 40-45°C) to apporeimately 12 ml. The tube is mix rapidly by inverting the tube twice. Poured directly on pre-made TM plates and incubated at 37°C for 3-8 days.
Mammalian cells
Cell culture and reagents
U2OS cells were kindly provided by The Danish Cancer Society. U2OS cell line is derived from the bone tissue of a patient suffering from osteosarcoma. U2OS cells show epithelial adherent morphology.
The cells are cultivated in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Penicillin, Streptomycin, and 10 % heat-inactivated foetal calf serum (FCS). Penicillin and Streptomycin are added to prevent any microbial growth. FCS is added to supply essential non-defined components, such as serum proteins and lipids. Supplemented DMEM medium is referred to as complete DMEM throughout the report. The cells are adherent and are kept in 75 cm2 culture flask until 80-100% confluency, where they are passed on to new culture flasks or cover slips.
Medium
- 500 ml DMEM
- 50 ml Fetal Calf Serum (FCS)
- 5 ml Penicillin
- 5 ml Streptomycin
Add FCS, penicillin, and streptomycin to a new flask of DMEM. Store at 5°C.
Cultivation of cells
25 cm2 culture flask
Complete DMEM
- Place the appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells (about 1-2 hours before).
- The U2OS cells are kept at -80°C until defrosted gently at 37°.
- Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette.
- The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded.
- The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask.
- The cells are kept at 37°C in a 5% CO2 incubator until the following day, where they are passed on to larger culture flasks.
Passing and maintenance of U2OS cells
Procedure for cells in 75 cm2 culture flask:
75 cm2 culture flask
50 ml vial
1, 2, 5, 10, 25 ml pipettes
Complete DMEM medium
0.05 % EDTA-trypsin
- The appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells.
- The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use.
- 1 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly.
- 1 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2.
- 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them.
- 2,5 ml (depends on the concentration of the cells) cell suspension is transferred to a new 75 cm2 culture flask.
- The cells are kept at 37°C in a 5% CO2 incubator until they are 80-100% confluent. This takes about 2-3 days. Then they are passed on to a new flask or plate.
Transferring the cells to coverslips
- The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use.
- 1,5 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly.
- 1,5 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2.
- 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them.
- Calculate the concentration of cells you want in the wells
- Cover slips are placed in the bottom of a 6-well plate (2 cover slips/well). 2 ml cell suspension is added gently to each well.
- The plate is placed in the incubator at 37C and 5% CO2 O/N.
Transfection of cells
- Disinfect LAF bench and gloves with ethanol before and after working in the LAF bench.
- To prepare the transfection, transfer 46μl optimem to a 1,5ml eppendorf tube per tranfection.
- Add 3 μl of Fungene 6 directly into the optimem and pipet up and down to mix.
- Flick gently on the tube to obtain further mixing
- Incubate for 5 mn at room temperature
- Add 1 μl of plasmid DNA directly into the optimem/Fugene mixture and pipet up and down to mix.
- Flick gently on the tube to obtain further mixing.
- If there is any liquid remaining on the side of the tube, spin very shortly in the centrifuge.
- Incubate the eppendorf tubes for 15 min at 37C.
- Take out the 6 cm dish with HEK293 cells from the incubator.
- In the LAF bench, add the 50 μl transfection mixture to the cells. Do it drop-wise into the medium.
- Use rocking motion to gently mix and place the dishes back in the incubator.
Coverslides for microscopy
- Delute PBS with MilliQ ten times, having final amount of 500ml.
- Sterile move the coverslips to a 24-well plate.
- Wash twice with PBS.
- Add 400 μl Formaldehyde to each well under the fume hood.
- Incubate for 12 min at RT. Remove liquid to the waste bin
- Wash three times with PBS.
- Remove coverslips and immerse shortly in MilliQ water before laid on lint-free paper for drying.
- Take 4 μ Vecta shield and place on cover slide. Repeat for each coverslip.
- When the coverslip is dry, place on coverslide with cells downwards.
- Fixate the coverslips with transparent nail varnish.
Purification of plasmids
EndoFree Plasmid Maxi kit from QIAGEN was used to purify plasmids for use in mammalian cells.
QIAGEN protocol
- Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for ~8 h at 37°C with vigorous shaking (~300 rpm).
- Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 100 ml medium, and for low-copy plasmids, inoculate 250 ml medium. Grow at 37°C for 12–16 h with vigorous shaking (~300 rpm).
- Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. Resuspend the bacterial pellet in 10 ml Buffer P1
- Add 10 ml Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 min. The lysis reaction time must not exceed 5 min.
- Add 10 ml chilled Buffer P3 to the lysate, and mix immediately but gently by inverting 4–6 times. Proceed directly to next step. Do not incubate the lysate on ice.
- Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 min. Do not insert the plunger!
- Remove the cap from the QIAfilter Cartridge outlet nozzle. Gently insert the plunger into the QIAfilter Maxi Cartridge and filter the cell lysate into a 50 ml tube.
- Add 2.5 ml Buffer ER to the filtered lysate, mix by inverting the tube approximately 10 times, and incubate on ice for 30 min.
- Equilibrate a QIAGEN-tip 500 by applying 10 ml Buffer QBT, and allow the column to empty by gravity flow.
- Apply the filtered lysate from step 9 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
- Wash the QIAGEN-tip with 2 x 30 ml Buffer QC.
- Elute DNA with 15 ml Buffer QN.
- Precipitate DNA by adding 10.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant.
- Wash DNA pellet with 5 ml of endotoxin-free room-temperature 70% ethanol (add 40 ml of 96–100% ethanol to the endotoxin-free water supplied with the kit) and centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet.
- Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of endotoxin-free Buffer TE.
"To determine the yield, DNA concentration should be determined by both UV spectrophotometry and quantitative analysis on an agarose gel."
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