CHAPTER 3. METHODS
3.1 M OLECULAR BIOLOGY METHODS
Genes of interest were amplified from Plasmodium falciparum 3D7 genomic DNA, DNA libraries, isolated plasmids or synthesized genes using Phusion High Fidelity DNA Polymerase (NEB) which has proof-reading activity to avoid undesired mutations. Primers and templates used for every construct can be found in Appendix 1. PCR conditions and reagents are detailed in tables below. Amplification was performed in a final volume of 50 µL. In those cases where no product was obtained or unspecific products were detected, the annealing temperature was optimized using a temperature gradient. Extension time was dependent on the product length and complexity.
PCR standard mix Phusion Polymerase
Reagent Volume(µl)
Final concentration
dH20 32.4 -
5X Phusion Buffer 10 1X
dNTP´s (10 mM) 5 1 mM
Primer forward (10 µM) 1 0.2 µM
Primer reverse (10 µM) 1 0.2 µM
Template 0.3 <250 ng
Phusion Polymerase (2U/µL) 0.3 0.012 U/µL
Thermocycling conditions for PCR with Phusion polymerase
PCR-step
Temperature (°C)
Time Cycles Initial denaturation 98 1 min
Denaturation 98 30 s
30 X
Annealing 48-65* 90 s
Elongation 72 1-3 min**
Final hold 4 ~
* Optimized depending on primer length and product complexity
**variable depending on amplicon size and template concentration
3.1.2 Purification of PCR products and digested vectors
Amplicons and restricted plasmids or inserts were purified from PCR and digestion reactions respectively using a commercial kit Nucleo Spin Gel and PCR Clean –up (Macherey- Nagel) according to manufacturer’s protocol. This guarantees the removal of enzymes (digestion enzymes and polymerases), oligonucleotides, salts and additives and the recovery of up to
~15µg DNA from 50 bp to at least 20 kbp.
3.1.3 Digestion of PCR products and vectors
To generate cohesive ends purified PCR products and vectors were digested with the respective enzymes in a volume of 100 µl with a final enzyme concentration of 0.02 U/µl.
Restriction enzymes used for every construct are listed in Appendix 1. Reaction was performed for 1-3 hours at 37°C except for construct SBP1mDHFRGFP-PHmut where a BstBI enzyme only active at 65°C was used. DNA integrity and correct digestion pattern was verified by agarose gel electrophoresis. Digested inserts and plasmids were purified as described above (3.1.2)
3.1.4 DNA ligation
To promote join of cohesive ends between insert and restricted vector a ligation reaction was performed using a molar ratio of 1:3 vector to insert. In a total volume of 10 µl, ~100 ng digested vector were incubated with the respective amount of insert, 1 μL 10x T4 ligase buffer and 400 U T4 ligase (NEB) for 1 hour at room temperature or overnight at 16 °C.
3.1.5 Screening PCR to detect bacterial clones
To screen for bacterial colonies carrying the desired plasmid after transformation (See 3.2.2) a PCR master mix with FIREpol polymerase was prepared using a vector specific primer and an insert specific primer following the composition outlined below. The screening PCR reaction was performed in a volume of 10 µl. From the LB-Amp plate where the transformed cells were smeared, a single colony was picked with a pipet tip, inoculated in a second plate to generate a master plate and placed in a PCR tube containing the master mix. At least 16 colonies were screened for every construct. Thermocycling conditions for screening PCR are detailed above. PCR reactions were analyzed by agarose electrophoresis to identify clones with the expected product size.
PCR standard mix (FIREPol Polymerase)
Reagent Volume(µl)
Final concentration
dH20 6.5 -
10X FIREPol Buffer 1 1X
dNTP´s (10 mM) 1 1 mM
Magnesium chloride (25 mM) 1 2.5 mM Primer forward (10 µM) 0.2 0.2 µM Primer reverse (10 µM) 0.2 0.2 µM FIRE Polymerase (2U/µL) 0.1 0.02 U/µL
Thermocycling conditions for screening PCR (FIREPol)
PCR-step
Temperature (°C)
Time Cycles Initial denaturation 95 2 min
Denaturation 95 40 s
30 X
Annealing 48 90 s
Elongation 68 1-3 min*
Final hold 4 ~
*variable depending on amplicon size and template concentration
3.1.6 PCR to verify genome integration
PCR verification of genome integration in P. falciparum was performed with FIREpol polymerase on genomic DNA (gDNA) of integrant cell lines and 3D7 in a final volume of 40 µl according to mix composition detailed below. Three different primer combinations were set up to confirm 5´ and 3´ integration and disruption of endogenous locus using 3D7 gDNA as control.
5´ integration was checked using a primer specific for the region upstream of the modified locus and a vector specific reverse primer. 3` integration was verified using a reverse primer specific for the downstream 3´ region and a vector specific forward primer.
Reagent Volume(µl)
Final concentration
dH20 25.5 -
10X FIREPol Buffer 4 1X
dNTP´s (10 mM) 4 1 mM
Magnesium chloride (25 mM) 2.4 1.5 mM Primer forward (10 µM) 1.6 0.4 µM Primer reverse (10 µM) 1.6 0.4 µM FIRE Polymerase (2U/µL) 0.4 0.02 U/µL
Template (gDNA) 0.5 -
PCR-step
Temperature (°C)
Time Cycles Initial denaturation 90 1 min
Denaturation 90 20 s
30 X
Annealing 41 20 s
Elongation 61-65* 1-4 min**
Final hold 4 ~
* Optimized by temperature gradient
**variable depending on amplicon size and template concentration
3.1.7 Sequencing of plasmids
To exclude mutations in isolated clones at least 0.8 µg of a purified plasmid were premixed with a specific primer (20 pmol) and adjusted with water to a final volume of 15 µl. Primers for sequencing should be ~ 20 base pairs and have binding site 30-60 nucleotides upstream of the start sequence. Samples were shipped to SeqLab (Sequence Laboratories Göttingen).
3.1.8 Agarose gel electrophoresis
DNA fragments (PCR products and digestions) were separated by length in an electric field in an agarose matrix (Garoff and Ansorge 1981). Agarose (Invitrogen) was dissolved in buffer 1X TAE to a final concentration of 1% and cooked in a microwave until agarose melted completely. Ethidium bromide was added to a final concentration of 1 µg/ml and agarose solution was poured in a gel tray and cooled down until it became solid. Agarose gel was placed in an electrophoresis chamber filled with 1X TAE. Samples were diluted with 6x DNA
loading dye and load into wells next to a DNA ladder to determine size of bands. Separation was performed for 25-30 minutes at 150V.
3.1.9 DNA precipitation
For P. falciparum transfection protocol 50-100 µg DNA are required. A volume of isolated plasmid (Midi Preparation) containing 50-100 µg DNA was mixed thoroughly with a 1/10 volume sodium acetate 3M, pH 5.0 and further precipitated with three volumes of absolute ethanol. Precipitated DNA was stored overnight at -20 °C before transfection procedure.
3.1.10 Isolation of genomic P. falciparum DNA
To verify genome integration, 5-10 ml culture of a P. falciparum transgenic cell line with a parasitemia 5-10% were harvested and centrifuged at 1800 rpm for 5 minutes. Supernatant was discarded and culture pellet was processed with the kit QIAamp DNA Mini Kit according to manufacturer´s protocol. Genomic DNA was aliquoted and stored at -20 C.