2 Material and Methods
2.2 Methods
2.2.1 Molecular biological methods
2.2.1.1 Polymerase chain reaction (PCR) for DNA amplification
Polymerase chain reaction (PCR) (Mullis and Faloona, 1987) was used to specifically amplify DNA fragments from DNA templates. For preparative PCRs Phusion polymerase with proofreading function was used. FirePol polymerase was used for colony screen and analytical PCR control (see next section). Specific oligonucleotides (primer pairs, forward and reverse flanking the 5’ and 3’ regions) were used from a 100 µM stock solution. Primers were designed using sequence information given on PlasmoDB (www.plasmodb.org). Typical PCR-reactions were prepared as follows:
Preparative PCR:
5x Phusion buffer 10 l dNTPs (2.5 mM) 5 l
Primer sense 1 l
Primer reverse 1 l
Template DNA 0.3 – 0.5 l
Phusion polymerase 0.3 l
dH2O to 50 l
PCR program:
Phase Temperature Time
Denaturation 95 C 4 min
Denaturation 95 C 15 s
30 cycles Annealing 42 C 30 s
Elongation 62 C x min
Storage 4 C
x depends on the length of the PCR product and was usually set to 1 min / 1000 bp. PCR settings were adjusted depending on the PCR product and primer-sequence.
2.2.1.2 Analytical PCR control and colony screen
Analytical PCR was performed to screen for positive clones after transformation into E.coli bacteria (colony PCR) or to check for correct integration. For the colony screen single colonies were picked from agar plates (over night cultured E.coli cultures after transformation) and used as template in a typical PCR-reaction as follows:
Analytical PCR:
10x FirePol buffer 1 l
MgCl2 1l
dNTPs (2.5 mM) 1l
Primer sense 0.4 l
Primer reverse 0.4 l Template DNA
/ bacterial colony
0.3 l
Phusion polymerase 0.1 l
dH2O to 10 l
PCR program:
Phase Temperature Time
Denaturation 95 C 4 min
Denaturation 95 C 30 s
25 cycles Annealing 45 C 30 s
Elongation 64 C x min
Storage 4 C
x depends on the length of the PCR product and was usually set to 1 min / 1000 bp. PCR settings were adjusted depending on the PCR product and primer-sequence.
2.2.1.3 Agarose gel electrophoresis
According to their length DNA fragments can be separated by agarose gel electrophoresis as based on their negatively charged phosphate backbone they will move toward the anode in an electric field. Samples were loaded onto 1 % agarose gels.
Agarose was mixed with 1x TAE buffer and dissolved by boiling. After cooling down, the agarose solution was transferred into a gel tray and Ethidiumbromide was added to a final concentration of 1 g/ml. To generate pockets for DNA loading combs were placed into the gel tray right after. The hardened gel then was transferred into an electrophoresis chamber filled with 1x TAE. Samples were mixed with 6x DNA loading dye and loaded into the pockets. Electrophoresis was performed at 120 V for 20 minutes.
1kb GeneRuler was used as a DNA ladder and separated bands were visualized via UV light using the ChemiDoc XRS+ system.
2.2.1.4 PCR-product purification
Obtained PCR-products and digested DNA fragments as well as vectors were purified using the NucleoSpin Gel and PCR Clean-up kit according to the manufacturer’s protocol and eluted in 20 - 40 l dH2O.
2.2.1.5 “Preparative” DNA digestion
Purified DNA fragments and plasmids were digested to create “sticky ends” for subsequent ligation. Depending on the used vector different restriction enzymes were used. DpnI was used if PCR-products were amplified from methylated template DNA.
The incubation time for preparative restriction was 2.5 - 3 h at 37 C.
Reagents Volume
10x NEB cut smart buffer 5 l
Each restriction enzyme 0.5 l
DpnI (for PCR-products only) 1 l
DNA (plasmid / PCR) 2 l / whole volume
dH2O ad 50 l
2.2.1.6 Analytical DNA digestion (test digest)
Plasmids were analytically digested using 2 µl of purified DNA (mini or midi DNA) in a total reaction volume of 10 µl. 1 µl of 10x cut smart buffer and 0.2 µl of diluted enzymes were added and adjusted to the final volume by addition of dH2O. After 1 h incubation at 37 °C the fragments were visualized by gel electrophoresis.
2.2.1.7 Ligation
Digested vector DNA and PCR-fragments were ligated using T4 ligase. Ligation was used to produce plasmids that can be transformed into E. coli for multiplication on LB-agar-plates. The ligation reaction mix was incubated for 20 minutes at room temperature.
Reagents Volume
10x T4 ligase buffer 1 l
PCR product 7 l
Vector DNA 1 l
T4 ligase 1 l
2.2.1.8 DNA ligation via Gibson assembly
Alternatively DNA ligation can be performed via Gibson DNA assembly (Gibson et al., 2009). This method allows the ligation of up to 6 inserts into one vector. This protocol does not need sticky ends but needs to overlap with the vector sequence in 15 - 35 bp.
Therefore only DpnI digestion is necessary. The ligation reaction was incubated for 60 minutes at 50 °C. A typical reaction is shown below:
Reagents Volume
Assembly master mixture 7.5 l
PCR product 1 l
Vector DNA 1 l
dH2O ad 10 l
2.2.1.9 Production of chemically competent E. coli
The rubidium chloride method was used to render E. coli bacteria chemo-competent (Hanahan, 1983). This treatment leads to a decreased stability of the bacterial cell wall resulting in an increased plasmid uptake. Therefore 20 ml LB medium were inoculated with a glycerol stabilate of the E. coli XL Gold strain and incubated at 37°C, overnight, shaking. 10 ml of the culture were transferred into an 200 ml LB-holding Erlenmeyer flask and incubated at 37°C, shaking, until an OD600 of 0.5-0.6. The bacteria were harvested by centrifugation af 2400 x g for 20 minutes at 4°C. Subsequently the pellet was re-suspended in 60 ml TFBI buffer and incubated on ice for 10 minutes. After another centrifugation step at same conditions the pellet was re-suspended in 8 ml TFBII buffer and 100 l aliquots were prepared in 1.5 ml reaction tubes and stored at -80°C until further use.
2.2.1.10 Transformation into chemically competent E. coli cells
For the transformation of the ligation reaction mix into chemically competent cells 50 µl aliquots of E.coli cells were thawed on ice for 5 - 10 minutes. The ligation mix (or 2 l of a sequenced plasmid) was added and incubated on ice for 10 minutes. After a 40 second heat shock at 42°C and following cool down on ice for 1 minute, 1 ml of LB medium (without ampicillin) was added and incubated at 37°C for 30 minutes, shaking, 750 rpm.
The suspension subsequently was evenly distributed on ampicillin containing LB-agar plates using sterile glass beads and cultured over night at 37 °C.
2.2.1.11 Overnight culture of E. coli for subsequent plasmid DNA preparation
For plasmid preparations bacteria from an agar plate (or from a glycerol stock) were inoculated in LB medium at 37 °C overnight with vigorous shaking. Volume was dependent on mini or midi preparation. For plasmid mini preparation 2 ml LB medium was used in a 2 ml reaction tube, plasmid midi preparation were inoculated in 150 - 200 ml LB medium using an 1 l Erlenmeyer flask.
2.2.1.12 Freezin of E. coli
For long term storage 500 µl overnight E. coli culture were resuspended in 500 µl of glycerol in a 2 ml reaction tube and stored at -80 °C.
2.2.1.13 Plasmid-DNA isolation (mini and midi preparation)
Plasmid-DNA was extracted from 2 ml overnight cultures using the NuleoSpin Plasmid Kit for mini preparation according to the manufacturer’s protocol. Midi preparations were purified from 150 ml overnight cultures using the QUIAGENPlasmid Midi Kit. In brief, these preparations are based on the principle of alkaline lysis of cells and subsequent binding of the DNA to a silica membrane under physiological conditions.
Mini preparation was eluted in 50 µl dH2O and midi preparations in 200 µl TE buffer.
Eluted DNA was used for restriction digest (mini preparation) or for transfection of P.
falciparum (midi preparation).
2.2.1.14 Determination of DNA concentration via Nanodrop
DNA concentration was determined using Thermo Scientific NanoDrop 2000c Spectrophotometer measuring the absorbance at 260/280 nm (A260/280). Hereby 260 nm is the absorbance maximum of nucleic acids, 280 nm the maximum of proteins. The
optimum A260/280 value for pure DNA is considered about 1.8. dH2O or TE buffer were used as blank depending on the elution solution. Software: NanoDrop 2000.
2.2.1.15 Sequencing of plasmid DNA
To verify correct integration of the PCR fragment into the vector and to exclude mutations of the insert purified plasmids were sent for sequencing. A sequencing primers (2 µl) that binds the vector in front of the insert was mixed with 5 µl of the plasmid DNA and dH2O was added to a reaction volume of 15 µl. Sequencing was performed by Sequence Laboratories (Seqlab, Göttingen).
2.2.1.16 DNA precipitation
For DNA precipitation 100 µg of purified plasmid DNA was mixed with 0.1 volume of sodium acetate 3 M pH 5.6 in a 1.5 reaction tube. Three volumes of 100 % ethanol was added, mixed by vortexing and stored at -20 °C overnight or at least for 20 minutes. The solution was centrifuged at maximum speed for 15 minutes. After removing the supernatant the DNA pellet was washed with 70 % ethanol. Following a subsequent centrifugation step the supernatant was removed and the pellet air-dried under sterile culture conditions and solved in sterile TE buffer (the volume was depend on the transfection method).