Genomic Isolation Protocol

Cardiac puncture of the mice was performed and the blood was collected in an heparinized tube.
After this 5ml PBS was added along with 75µl saponin which was left for 5 minutes at RT. The
blood was then divided into two tubes of 2ml and then it was centrifuged it at 12000rpm for
5mins at room temperature and washed with 1ml PBS solution thrice with centrifuging it at the
same rpm and the last PBS wash was done with 20µl proteinase k. After this 200µl cell lysis
buffer was added from the Promega kit which was kept on the hot plate at 56ºC along with
vortexing it at intervals of 10-15 mins. After this 250µl binding buffer from the same kit was
added, the tube was vortexed. It was now poured in a collection tube and centrifuged at
maximum speed for 1 min. Now 500µl of washing buffer was added and it washed twice after
which a 1 min spin at max speed was performed. Now we kept the eppendorfs in the dry bath for
30 mins to 1 hour and then elute in 30-50µl of NFW.

Retro-orbital Blood transfer

Blood smear of the mice was made, stained with Giemsa staining solution and then parasitemia
count of an average of 10 fields was performed. After the calculations the next step was to
collect blood from mice through its retro-orbital sinus using a heparinized Pasteur pipette and the
dilutions were made accordingly. After the dilutions were made the blood sample was injected
intravenously in three healthy mice, for each sample.

Immunisation protocol
First mosquito washing was done with DMEM thrice, 1 min each wash in ice. Then the
dissection of the salivary glands of the mosquito was performed, once the salivary glands were
collected in an eppendorf DMEM is poured in it and it is plunged using an ethanol wiped
plunger to burst open the salivary glands so that the sporozoites move freely in the solution.
After each set of plunging, we gave it a short spin for 15 seconds. Then a dilution of 10X was
made (dilution of 10X or 50X can also be made according to the number of sporozoites). 10µl of
the dilution was transferred to the hemocytometer for sporozoite count, before which it is kept on
the wet chamber for the dilution in the hemocytometer to settle down. While dissecting the
mosquitoes the number of female mosquitoes was counted.

The total number of sporozoites was calculated using the dilution factor and the sporozoites per
mosquito was enumerated using the following formula:

Sporozoites per mosquito= Sporozoites per µl × Total volume/ Total number of mosquitoes
Drug Cover
After every immunization the mice were kept under drug cover of Chloroquine drug in the
amount of 0.8mg/mice. The dilution was made as follows:

35* 0.8 = 28mg

28mg Chloroquine was dissolved in 7ml DI.