The first step done when cloning a new sensor was to do virtual cloning using SECentral (Scientific & Educational Software, NC, US). This consisted of the loading of a vector map for the sensor to be cloned and a vector map for the backbone to clone in to. Then finding proper cloning sites by searching the multiple cloning site (MCS) for sites that uniquely opened the backbone and cut on both sides of the sensor. In most cases no such sites were found and the strategy then turned to adding new cloning sites using polymerase chain reaction (PCR). For this purpose two sets of primers were designed in SECentral. The first set served as templates for the insertion of new restriction sites through overhangs. The second set was designed as sequencing primers of∼8bp that bound∼200bp upstream of the insert and the same down-stream (reverse direction). Since PCR is error prone, these sequencing primers were used for sequencing of the cloned plasmid to ensure integrity of the original sequence. When design of restriction sites were necessary, the design was made in such a way as to ensure that the Kozak consensus sequence, CCACC(ATG)(Kozak, 1986), was present between the restriction site and the cDNA start codon of the insert. As a reference, most procedures were done according to those described in “Molecular Cloning: A Laboratory Manual” (Sambrook and Russell, 2001). All restriction nucleases used were from New England Biolabs.
2.5.1 PCR amplification
PCR amplification of cDNA sequences of interest was carried out on a PTC-150 MiniCycler thermocycler. Initially an optimization PCR was carried out with different (0, 1.5 and3mm) magnesium chloride (MgCl2) concentrations and different annealing temperatures (between 60 and72℃ in order to establish optimal conditions. While most sensors have similar size and are composed of mostly fluorescent proteins which are very similar in structure, it was quickly noticed that this step for most cloning could be skipped. If nothing special was required3mm MgCl2and an annealing temperature of72℃ was used. 50µL PCR reaction mix then contained:
1. 27µL millipore H2O
2. 10µL reaction buffer (Phusion HF 5x) 3. 1µL backbone template (5ng µL−1) 4. 5µL forward primer (4pmol µL−1) 5. 5µL reverse primer (4pmol µL−1)
6. 1.5µL dNTP mix (dATP, dCTP, dGTP, dTTP10pmol µL−1) 7. 0.5µL Phusion polymerase (2U µL−1)
The thermocycler was programmed according to the following:
initial separation 30s at 98℃
separation 30s at 98℃ annealing30s
at 60-72℃ elongation 30s at 72℃
cooling ∞at4℃ 25x
Meaning that separation, annealing and elongation was done for the given time at the given temperature and that it was repeated 25 times. The last step meant that the PCR reaction was cooled to4℃ after finishing and could thus be run over night if needed.
After amplification the reaction was analyzed for the correct size with agarose gel elec-trophoresis (see section 2.5.3).
2.5.2 Restriction of DNA
Restriction of DNA was done according to the enzyme manufacturers (New England Biolabs) intructions. Enzymes and DNA were mixed in the correct buffer and incubated in water bath at the recommended temperature for60min. Restriction was done in a total volume of50µL with5µg plasmid backbone or∼1µg PCR reaction,5µL buffer and1.5U restriction enzyme per µg DNA. In the case that the temperature and activity in the same buffer was at least 75% enzymes for both restriction sites were both added to the reaction. If the temperatures did not match, incubation with the enzyme with lower optimal temperature was done first, the second enzyme then added and the temperature raised to the new optimal. If the buffers had to be switched the reaction was first restricted with one enzyme, purified using a PCR purification kit (Qiagen) and then prepared again with the second enzyme in the new buffer and incubated again.
2.5.3 Agarose gel electrophoresis
Agarose gel electrophoresis was used to analyze the fragments from DNA restriction digests for purity, quantity and size. The gel was prepared by weighing the proper amount of agarose, dissolving it in 1x TBE buffer by heating in a microwave oven. When completely dissolved, the bottle was held under cold flowing water for about one minute to reduce the time taken for polymerization. 1µL ethidium bromide per50mL solution was then added, mixed and poured into a gel tray. A lane comb was inserted and the gel left to polymerize for∼30min at room temperature. The amount of agarose was chosen based on the size of the fragment of interest. For fragments smaller than∼500bp a 2% gel was used. For fragments between
∼500bp and∼1000bp 1.5% and for fragments larger than∼1000bp 1%. The gel tray was put in the gel chamber and 1x TBE was added to cover the gel. Samples were mixed with DNA loading buffer (6x) and TE to a final volume appropriate for the wells of the gel and pipetted in the wells. The gel chamber was closed and connected to a power supply which was set
to deliver direct current of80V and left to run until sufficient separation of the fragments of interest had occurred. The DNA bands in the gel was then visualized under UV-illumination on a Gel Documentation 2000 UV-transilluminator with a camera connected to a computer running Quantity One (BioRad) software.
2.5.4 DNA production, extraction and purification
In cases were the DNA from a agarose gel electrophoresis was to be further used, the band was cut out under low-intensity UV illumination and purified using a QIAquick Gel Extrac-tion Kit according to the manufacturers instrucExtrac-tions. For mini preps, extracExtrac-tion of DNA from lysed bacteria was done using a QIAquick Mini Prep Kit according to the manufacturers in-struction. The DNA was eluted in36µL10mm Tris-buffer of which 6µL could be used to determine the concentration. The purity of the sample after extraction with either of these two kits was high enough that no further purification such as precipitation was necessary for sequencing, ligation or transformation of bacteria.
To obtain plasmid DNA in milligram quantities, a mega-prep was done using a Macherey-Nagel NucleoBond PC2000 kit on two liters of bacterial culture cultivated over night from a 1mL mini-prep aliquot. The procedure was the same as the one given in the manufacturers manual with the exception that the column was used twice. After the first run through and elution the column was washed with the right buffer and the second half of the batch was loaded. The plasmid DNA extraction procedure consists of the following steps:
1. pelleting of bacteria by centrifugation
2. resuspension in resuspension buffer supplemented with 100µg ml−1 Rnase A to de-grade
3. alkaline lysis of bacterial cells (1% SDS,10mm EDTA,200mm NaOH)
4. precipitation of SDS, proteins and protein associated DNA and removal by centrifuga-tion (2.8m KAc, pH 5.1) supernatant contains plasmid DNA
5. binding to a pre-equilibrated anion-exchange resin column
6. washing of the column to remove chromosomal DNA and proteins (100mm Tris-HCL, 15% ethanol,1.15m KCl, pH 6.3)
7. elution (100mm Tris-HCL, 15% ethanol,1m KCl, pH 8.5)
After elution, plasmid DNA from large scale production was concentrated and desalted first by isopropanol precipitation and subsequently again by ethanol precipitation. Finally the pellet from ethanol precipitation was resuspended in 1x TE buffer over night.
2.5.5 DNA concentration determination
The concentration of DNA was determined by measuring the optical density on a spectrom-eter at260nm. A ratio of the optical density measured at 260 and 280nm of more than 2 was taken as indicative of low protein contamination of the DNA sample. In cases were the DNA-concentration was to low to be recorded on the spectrometer, the concentration was assessed on a agarose gel. In this case, a λHindIII digest was used as reference. Bands in the sample of similar size to those in the standard was compared to the intensity of the bands in the standard. Those that most closely matched in intensity was assumed to have the concentration of the corresponding band in the standard.
2.5.6 Ligation and transformation of bacteria
The restricted backbone and insert was ligated and transformed into bacteria for growing and selecting of clones. First the concentration of restricted backbone and insert was assessed as described in section 2.5.5. Then a total amount of between∼150-250ng backbone and insert was mixed in a 1:3 molar ratio together with T4 ligation buffer and2000U T4 DNA ligase with sterile water added to a final volume of20µL. The ligation reaction was left to react at room temperature for20min. To control for religation events of the backbone due to insufficient separation or purification from gel extraction, a second ligation was set simultaneously with the first were the insert DNA was not added. After ligation, the reactions were put on ice before being used to transform bacteria.
For transformation, electroporation cuvettes were pre-cooled on ice. 70µg electrocompe-tent bacteria suspension was thawed on ice. DH5α E.coli cells (ElectroMAX) was used when pre-cloning of received vectors had to be done. Otherwise, since all cloning was done in or-der to create pAAV vectors, electroporation-competent cells, “stop unwanted rearrangement events” (SURE) E.coli cells were used in order to reduce the number of clones with corrup-tion of ITR sequences in the plasmid genome. 4µL ligation reaction was transferred to the vessel containing the E.coli, mixed carefully and left for 1min on ice. The full volume of bacteria/ligation mix was then transferred to the electroporation cuvette. The outside of the
cuvette was quickly wiped with a paper napkin to remove ice and moisture. The cuvette was then placed in the electroporator (Bio-Rad Gene Pulser II) and flashed with1.8kV potential, 200Ω resistance and25µF capacitance. Immediately after flashing, 800µL SOC++ medium was added to the cuvette, carefully mixed and transferred to a1.5mL eppendorf tube. The procedure was repeated for all ligation reactions after which the tubes with the flashed bac-teria suspension were incubated at37℃ with vigorous shaking for45min to let them recover and initiate expression of the antibiotics resistance protein encoded for by the plasmid before being exposed to antibiotics.
For plating, LB agar plates with the proper antibiotics (e.g. ampicillin100were pre-warmed to room temperature and200µL bacteria suspension plated with the help of a bent Pasteur pipette. For difficult ligations, the rest of the bacteria suspension was centrifuged shortly, 300µL supernatant removed, the rest resuspended and plated on a second agar plate. The same was done with any control reactions. The plates were then incubated at37℃ over-night for at least 15 hours or until colonies were easily seen by the naked eye.
2.5.7 Picking of clones and verification of plasmid
The number of clones on LB agar plates from control ligation were compared with the proper ligation. In most cases the ratio of proper to control was> 100 and about 8 colonies were picked and cultivated in4mL LB media over night. From this,2mL was used with a QIAquick Mini Prep Kit (QIAGEN) (see section 2.5.4) to prepare DNA for checking plasmid integrity and sequencing of PCR insert fragment. 100ng from this preparation was restricted with SmaI restriction nuclease (NEB) and analyzed using agarose gel electrophoresis (see section 2.5.3) to verify the integrity of the ITRs of the AAV plasmid. A virtual restriction was done to infer the correct size of the bands from SmaI restriction and the bands on the gel was compared to these. Corrupted ITRs were evident as the wrong band from that expected due to SmaI not restricting in corrupted regions.
Since PCR amplification is an error prone process, the amplified fragment was sequenced in order to assure that it had the correct sequence. After mini-prep (section 2.5.4) and ITR in-tegrity verification, a sample was sent for sequencing with the company SEQlab. The service at SEQlab used was “extended hotshot 800” which sequences∼800bp in one stretch with high fidelity. SEQlab was supplied with600ng plasmid DNA and20pmol primer in 10mm Tris-HCL. For PCR products that were longer than800bp a second sample with a primer for the
reverse direction was used. For really long inserts, were sequencing in the forward and re-verse directions from the ends of the insert was not sufficient, additional sequencing primers were designed either from the vector map or if not at hand, from a previous sequencing step done from the ends of the insert.