We use two protocols to transfect linearised DNA constructs into bloodstream form T. brucei, depending on the integration target and the desired transformation efficiency – both rely on a nucleofection apparatus running progamme X-001 (Lonza)

Protocol 1: standard

This is particularly useful for transfecting pRPa constructs, which integrate at the ‘landing pad locus’ in 2T1 cells, or similarly efficient construct-target combinations

1. Digest 10 µg construct with the appropriate restriction enzyme(s) to generate a linear DNA molecule with termini to allow integration into the T. brucei genome by homologous recombination
2. Clean the digested construct by phenol:chloroform:isoamylalcohol extraction and ethanol precipitation (do not include any salts such as sodium acetate, as these will interfere with electroporation); resuspend the precipitated DNA in 10 µl sterile distilled water
3. Pellet 25 million bloodstream form T. brucei at 1000 g for 10 minutes
4. Pour off the supernatant, remove as much of the residual media as possible without disturbing the cell pellet, and resuspend in 100 µl cytomix (pre-warmed to 37°C) plus 10 µl DNA solution
5. Transfer cell-cytomix-DNA mixture to a 2 mm gap cuvette and apply nucleofection programme X-001
6. Transfer cells to 60 ml media and add appropriate selective drugs 4-6 hours later
7. Transfer to two 48 well plates: ‘neat’ and ‘5-fold diluted’; positive wells should be transferred to 10 ml cultures five days post-electroporation

Cytomix (filter sterilise and store at 4°C)

KCl	120 mM
HEPES pH7.6	25 mM
1K2HPO4/KH2PO4 pH7.6	10 mM
MgCl2.6H2O	5 mM
EGTA pH7.6	2 mM
CaCl2	150 µM
Glucose	0.5%
Albumin, bovine serum	100 µg/ml
2Hypoxanthine	1 mM

¹ 10x K₂HPO₄/KH₂PO₄ pH7.6: 8.66ml 1 M K₂HPO₄, 1.34 ml 1M KH₂PO₄, 90 ml H₂O (store at room temperature)
² 100x Hypoxanthine: 0.4 g NaOH, 100 ml H₂O, 1.36 g hypoxanthine (store at -20°C)

Recommended drug concentrations

Drug	Stock (mg/ml)	Selective (µg/ml)	Maintenance (µg/ml)
Phleomycin	10 mg/ml	2	1
G418	10 mg/ml	2	1
Puromycin	2.5 mg/ml	2	1
Hygromycin	5 mg/ml	2.5	1
Blasticidin	10 mg/ml	10	5

 

Protocol 2: nucleofection

We use this protocol to integrate cassetes at RNA polymerase II transcribed loci; these typically represent much less efficient integration targets, and this approach is generally used for transfecting disruption or native tagging constructs

1. As per Protocol 1
2.             -“-
3.             -“-
4. Pour off the supernatant, remove as much of the residual media as possible and resuspend in 82 µl human T cell nucleofector solution, 18 µl supplement 1 (Lonza; both at room temperatue) plus 10 µl DNA solution
5. Transfer cell-nucleofector solution-DNA mixture to a 2 mm gap cuvette and apply nucleofection programme X-001
6. As per Protocol 1
7.             -“-