Hasenkamp Transfection of P.
falciparum(Hasenkamp et al., MJ 2012)
The protocol is based on the Deitsch method of preloading erythrocytes with plasmid and
seems to be superior compared to any other method.
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Prepare 150 µl packed fresh erythrocytes (prewashed with 15 ml cytomix) in 300 µl
cytomix (150 cytomix without plasmid plus 150 µl packed erys)
Add 40 µg of the plasmid that is to be transfected in 100 µl cytomix and transfer to
cuvette, store on ice.
electroporate at 310 V, 960 µF. Add immediately 1 ml complete medium, transfer to
15 ml Falcon tube and wash cuvette with 1 ml, add ad 10 ml complete medium and
centrifuge at 1500 rpm.
take floated schizont stage parasites up in complete medium and add 2*108 purified
schizont stage parasites (this is 40 µl of floated parasites at 100% parasitemia!),
cultivate in 3% hematocrit, take less parasites if you don´t have that much, 10 µl
parasites works equally (or maybe better)
two days after transfection, add drug (WR99210 at 2,5 to 5 nM, or Blasticidin S at 2,5
µg/ml)
after 4-6 days all parasites should have vanished, change 30% of the blood.
without visible parasites change medium every two days
add blood every 4 days until the appearance of parasites
Cytomix Deitsch/Sá/Gölnitz protocol
There is no MgCl2 in this mix as is found in Sidhu/Wellems protocol. This cytomix can be
frozen and thawn afterwards – however, bringing salt precipitates in solution takes one night
at 37°C.
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100 mM KH2PO4 (MW: 136,09 g/l, for 50 ml 0,68 g)
100 mM K2HPO4 (MW: 174,18 g/l, for 100 ml 1,74 g)
1 M Ca2Cl (MW: 110,94 g/l, for 50 ml 5,54 g)
500 ml of cytomix:
7 ml
100mM KH2PO4
43 ml
100mM K2HPO4
75 l
1M Ca2Cl
3,25 g
KCl (MW: 74,5 g/l)
0,38 g
EGTA (MW: 380,4 g/l)
50 ml
HEPES (MW: 238,3 g/l): prepare 250 mM
HEPES at
pH 7.5 and use this solution,
establishing the
right pH afterwards using
salt will result in
hemolysis!
 shake and sterile filter 0,22 m