Molecular biological methods

2.2.3.1 Polymerase chain reaction (PCR)

Two different DNA polymerases were used for the amplification of a DNA template. For preparative PCRs, Phusion polymerase with a proofreading function was used. For diagnostic PCRs, FirePol polymerase was used. The oligonucleotides used are listed in 2.1.9. Typical PCR-reactions were prepared as follows:

Preparative PCR µl Diagnostic PCR µl

5x Phusion buffer 10 10x FIREPol buffer 1

dNTPs [2 mM] 5 dNTPs [2 mM] 1

Primer fwd [50 µM] 0.5 Primer fwd [50 µM] 0.4

Primer rev [50 µM] 0.5 Primer rev [50 µM] 0.4

Phusion® High-Fidelity DNA Polymerase (2 U/µl)

0.5 FirePol® DNA

Polymerase (5 U/µl)

0.1

Template [100 ng/µl] 1 Template/colony 0.2

dH2O 32.5 dH2O 5.9

MgCl2 [25 mM] 1

Phase Temperature Time

Denaturation 95 °C 4 min

25-30 cycles

Denaturation Primer annealing Elongation

95 °C 48-70 °C 64-72 °C

30 s 30 s X min

Storage (optional) 4 °C ∞

(X) depends on the expected size of the PCR-product and was usually 1 min per 1,000 base pairs (bp).

2.2.3.2 PCR-product purification

To purify PCR-products and digested vector DNA for subsequent ligation, the NucleoSpin Gel and PCR Clean-up kit were used, according to the manufacturer’s protocol. PCR products and vector DNA were eluted with a 15-30 µl AE elution buffer.

2.2.3.3 DNA restriction digest

Preparative digests of PCR products and vectors were performed using different DNA restriction enzymes to create sticky ends for the cloning of plasmids. Analytical digests were performed of mini and midi DNA preparations to exclude recombination and to confirm the correct insertion of PCR products into the plasmid. The incubation time for preparative digests was 2-3 h, and for analytical digests it was 30-60 min, each at 37 °C. Analytical digests were performed using at least five different enzymes, resulting in a distinct vector-specific band pattern of fragmented DNA within the agarose gel. Typical digest-reactions were prepared as follows:

Preparative digest µl Analytical digest µl

10x Cut Smart Buffer 2 10x Cut Smart Buffer 2

Restriction enzyme A [20 U/µl] 0.2 Restriction enzyme A [20 U/µl] 0.2 Restriction enzyme B [20 U/µl] 0.2 Restriction enzyme B [20 U/µl] 0.2 Vector/insert [100 ng/µl] 4 Restriction enzyme C [20 U/µl] 0.2

dH2O 13.6 Restriction enzyme D [20 U/µl] 0.2

Restriction enzyme E [20 U/µl] 0.2

Vector [100 ng/µl] 4

dH2O 13

After a preparative digest of a vector, 0.5 µl of CIP phosphatase was added to the reaction and incubated for 30 min at 37 °C in order to dephosphorylate the 5’ ends of digested vector DNA.

2.2.3.4 DNA fragment ligation

Digested PCR products and vectors were ligated using the T4 ligase. The ligation mix was incubated for 30-60 min at room temperature (RT) and heat-inactivated by incubation at 65 °C for 20 min. Afterwards the ligation-reaction was used for the transformation of chemo-competent E. coli cells. Needed volumes of the cut vector and the insert were calculated depending on their length. Generally, a vector/insert ratio of 1:3 was used. Given a vector length of 10,000 bp and an insert size of 1,000 bp, a ligation-reaction was prepared as follows:

Ligation µl

10x T4 ligase buffer 1

Vector [100 ng/µl] 0.5

Insert [100 ng/µl] 0.15

T4 ligase (400 U/µl) 0.5

dH2O 7.85

2.2.3.5 Agarose gel electrophoresis

For agarose gel electrophoresis, usually 1 % agarose gels were used in this study. The agarose was mixed with a 1x TAE buffer and dissolved by boiling using the microwave. Ethidium bromide was added to a final concentration of 1 μg/ml. The solution was transferred into a gel tray and combs were placed to create pockets to allow for the loading of DNA samples. After the solidification of the gel the tray was transferred to the electrophoresis chamber, which was filled with 1x TAE. The DNA samples were prepared by adding of a 6x DNA loading buffer in a 1:6 ratio and loaded into the pockets. Electrophoresis was performed at a voltage of 150 V for 15-25 min. The size of the DNA fragments was analyzed under UV light by comparison to a DNA ladder using the ChemiDoc XRS+ imaging system. Vectors subjected to preparative digests (see 2.2.3.3) were separated using a 0.5 % gel.

2.2.3.6 Colony PCR-screen

After the overnight incubation of agar plates containing transformed E. coli colonies, single colonies were analyzed to determine if they contained the desired vector and the new insert. For this, diagnostic PCR (see 2.2.3.1) was performed. Sterile pipette tips were used to transfer single colonies into the PCR reaction volume. Primers that bind within the new insert and the vector were selected. The resulting PCR products were analyzed using agarose gel electrophoresis.

2.2.3.7 Plasmid preparation

Plasmids were either purified with the Nucleo Spin Plasmid Kit for small-scale purification (1.8 ml of overnight culture, Mini) or with the QIAGEN^© Plasmid Midi Kit for the isolation of plasmids (Midi) used for P. falciparum transfection (see 2.2.6.6), according to the manufacturers’ protocols. Plasmids from Minis were eluted with a 20-30 µl TE buffer. Plasmids isolated with the Midi Kit were usually eluted with a 200 µl TE buffer.

2.2.3.8 Determination of DNA concentration

DNA concentration was determined using the Thermo Fisher Scientific NanoDrop 2000c spectrophotometer to measure the absorbance at 260 nm. The purity of the DNA is determined by the quotient of the absorption of DNA at 260 nm and of proteins at 280 nm (260/280 nm). The optimum 260/280 value for pure DNA is considered about 1.8. A value < 1.8 indicates contamination with proteins, while a value > 1.8 indicates contamination with RNA.

2.2.3.9 Sequencing of plasmid DNA

After the analytical digest, the insert was sequenced to confirm that it does not contain mutations introduced during the preparative PCR (see 2.2.3.1) amplification process. For the sequencing-reaction in a 1.5 ml reaction tube, a final vector concentration of 80 ng/µl in a volume of 15 µl was desired. The final sequencing primer concentration was adjusted to 10 µM. The sequencing was performed by Seqlab, Göttingen. A typical sequencing reaction was prepared as follows:

Sequencing µl

Vector [100 ng/µl] 12

Primer fwd/rev [50 µM] 3

2.2.4.0 Plasmid DNA precipitation for transfection

For DNA precipitation, 50 μg of purified plasmid DNA (Midi-isolated, see 2.2.3.7) was mixed with a 0.1 volume of sodium acetate and three volumes of 100 % ethanol in a 1.5 reaction tube and left at RT for 20 min. During gentle mixing, a cloudy DNA precipitate becomes visible. The solution was centrifuged at 16,000 x g for 15 min. After removing the supernatant, the DNA pellet was washed with 500 µl of 70 % ethanol. Following a subsequent centrifugation step, the supernatant was removed, and the pellet was air-dried under sterile culture conditions.

The opaque DNA pellet was resuspended in 10 µl of sterile TE buffer and subjected to transfection (see 2.2.6.6).

2.2.4.1 Isolation of genomic DNA from P. falciparum

After saponin lysis of parasites (see 2.2.6.7), gDNA was isolated using the QIAamp^© DNA Mini Kit according to manufacturer’s protocol. DNA was eluted in a 50-100 µl TE buffer.