Materials and Reagents

1. Eco Nitrile PF 250 Gloves (ecoSHIELD^TM)
2. Filter tips Low retention 1,000 µl, 200 µl, 20 µl (ClearLine, catalog numbers: 713118 , 713117 , 713115 , respectively)
3. 10 µl Extended Length Filter Tip (Neptune Scientific, catalog number: BT10XL )
4. Tissue culture flask 25 version “Vent” (TPP^®, catalog number: 90025 )
5. Vacuum Filtration 500 “rapid”-Filtermax, PES membrane 0.22 μm pore size (TPP^®, catalog number: 99500 )
6. Microcentrifuge 1.5 ml tubes (Corning, Axygen^®, catalog number: MCT-175-C )
7. Polypropylene conical 50 ml centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050 )
8. 2 ml serological pipettes (FALCON^®, catalog number: 357507 )
9. 5 ml serological pipettes (SARSTEDT, catalog number: 86.1253.001 )
10. 10 ml serological pipettes (SARSTEDT, catalog number: 86.1254.001 )
11. 25 ml serological pipettes (SARSTEDT, catalog number: 86.1685.001 )
12. Dulbecco's Phosphate-Buffered Saline (DPBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14040091 ) stored at 4 °C
13. Schneider’s Drosophila Medium w: L-Glutamine and 0.40 g/L NaHCO₃ (PAN^TM BIOTECH, catalog number: P04-91500 ) stored at 4 °C
14. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 ), sent at -20 °C, then heat-inactivated (see Recipes from Bio-protocol by Reverte and Fasel, 2019), aliquoted to 50 ml tubes and stored at -20 °C
15. HEPES Buffer 1 M (BioConcept, catalog number: 5-31F00-H ) stored at 4 °C
16. Penicillin-Streptomycin (P/S) solution (10,000 IU/ml P and 10 mg/ml S) (Bioconcept, catalog number: 4-01F00-H ), sent at -20 °C, then stored at 4 °C for short-term storage
17. Hemin BioXtra, from Porcine (Sigma-Aldrich, catalog number: 51280 ) stored at 4 °C
18. Folic acid (Fluka^TM, catalog number: 47620 ) stored at 4 °C
19. 6-Biopterin (Sigma-Aldrich, catalog number: B2517 ) stored at -20 °C
20. TRI^® reagent (Molecular Research Center, MRC, catalog number: TR118 , 500 ml) stored at 4 °C
21. Chloroform (Thermo Fisher Scientific, Fisher Chemical, catalog number: C/4960/15 ) stored at room temperature
22. Isopropanol (Thermo Fisher Scientific, Fisher Chemical, catalog number: P/7490/17 ) stored at room temperature
23. Ethanol absolute (Thermo Fisher Scientific, Fisher Chemical, catalog number: E/0650DF/17 ) stored at room temperature
24. UltraPure^TM DNase/RNase-free Distilled Water (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977-035 ), shipped at room temperature, stored at 4 °C before opening, aliquoted to 1.5 ml tubes and stored at -20 °C
25. DNase I, RNase-free 10 U/µl with an activity of 0.2 U/µg on calf thymus DNA (Roche, catalog number: 10 776 785 001 ) stored at -20 °C
26. Nuclease S1 (#E576) (Promega, catalog number: M5761 ) stored at -20 °C
    Note: Pay attention to the fact that the concentration is specific to each tube and is indicated on the tube by the manufacturer.
27. Buffer S1 (#E578) (Promega, catalog number: M577A ) stored at -20 °C
    Note: The old reference was at 7.4x, but now Nuclease S1 Reaction Buffer is provided at 10x.
28. 1 kb DNA Ladder 500 µg/ml (NEB, catalog number: N3232S ) stored at 4 °C
29. 6x Gel Loading Dye (Purple), no SDS (NEB, catalog number: B7025 ) stored at 4 °C; supplied with the 1 kb DNA Ladder
30. Standard Agarose–Type LE for routine gel electrophoresis (BioConcept Ltd, Amimed, catalog number: 7-01P02-R ) stored at room temperature
31. Tris(hydroxymethyl)aminomethane, Molecular biology (Biosolve, catalog number: 20092391 ) stored at room temperature
32. Boric Acid (Crystalline Powder/Electrophoresis grade) (Fisher BioReagents, catalog number: BP168-1 ) stored at room temperature
33. EDTA, Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (Crystalline Powder/Electrophoresis grade) (Fisher BioReagents, catalog number: BP120-1 ) stored at room temperature
34. SDS, Sodium Dodecylsulfate, Solution 20% pure (AppliChem PanReac, ITW Reagents, catalog number: A3942,1000 ) stored at room temperature
35. Ultra-pure water, type Ultra Clear^TM (BLANC-LABO) stored at room temperature
36. RNase AWAY^TM (Molecular BioProducts^TM, MBP, catalog number: 7003 ) stored at room temperature
37. SYBR^TM Safe DNA Gel Stain (Thermo Fisher Scientific, Invitrogen, catalog number: S33102 ) stored at 4 °C
38. Complete parasite culture medium (see Recipes)
39. Ethanol 75% (see Recipes)
40. 10x TBE (see Recipes)
41. 2 mM EDTA (see Recipes)
42. 1x TBE (see Recipes)
43. 1x TBE-SDS 2% (see Recipes)
44. 1 kb DNA Ladder (50 µg/ml) (see Recipes)
    

Equipment

1. Autoclaved glass bottles (SCHOTT DURAN)
2. -80 °C freezer
3. Forma^TM Steri-Cycle^TM CO₂ Incubator at 26 °C (Thermo Fisher Scientific, catalog number: 371 )
4. Pipette controller (INTEGRA, catalog number: 155017 )
5. Pipettes 1,000 µl, 200 µl, 20 µl (Gilson^®)
6. Pipette 10 µl (Discovery Comfort)
7. Laminar flow hood parasite culture (SafeFAST Premium)
8. Chemical hood
9. Centrifuge 5810 R (Eppendorf) at room temperature and set here at 24 °C
10. Centrifuge MICRO STAR 17 (VWR) kept in a cold room at 4 °C
11. NanoDrop^TM ND-1000 Spectrophotometer (ThermoFisher Scientific)
12. Water bath (JULABO GmbH)
13. Balance (NewClassic MF, MS4002SDR, METTLER TOLEDO)
14. Ultra Clear^TM (BLANC-LABO) system
    Note: The Ultra Clear^TM (BLANC-LABO) system delivers Ultra-pure DNase/RNase-free water.
15. Mini-gel migration system “home-made” (see Figure 2)
16. Electrophoresis Power Supply (GIBCO BRL, Life Technologies, ST305) and power cables
17. Microwave (FAR)
18. Blue LED transilluminator (Dark Reader DR-88X, Clare Chemical Research)
19. Camera (Computar iAi CVAA5 IR No C510390B RoHS)
20. Video Graphic Printer (SONY, UP-895CE)
    

Procedure