JIGGERS AND TUNGA PENETRANS DNA EXTRACTION PROCEDURE ONE Reagents 1. 10% SDS – NaCl extraction Buffer (ratio 1:1) 2. Chroloform: Isoamly alcohol (24:1) 3. Isopropanol 4. 70% Ethanol 5. Low salt T.E Material 1. Gravid Jiggers :- Sample 2. Eppendorf Tubes (size 2 ml capacity) 3. SOPs 4. Pestles 5. Acid washed sterile sand Equipment 1. Thermometer 2. Centrifuge;- Heraeus PICO 17 ( Thermo scientific) (SN; 42101629, Ref; 75002410) 3. Micro- Pipette (1-10ul)- GILSON (MC 28443) 4. Measuring cylinder 5. Fridge - LG (SN; 811TRWA00017) 6. Water bath -65 0 c – Improvised METHODOLOGY - Nine of the free moving fleas that were preserved in 70% ethanol were put in eppendorf tube and the tube was labeled. FS:- Flea SDS - In another tube, One Gravid jiggers that has been in a fridge for more than six months not in 70% ethanol were put and labeled; OS: - Old Jiggers SDS - In another tube, One Gravid Jigger (Smaller in size ), that had being preserved in 70% ethanol was added and the tube was labeled SS1:- Sterile Jigger SDS - Another one gravid jiggers (Larger in size ), that was preserved in 70% ethanol was also put into another tube and labeled SS2:- Sterile Jigger SDS - To all the four tubes, half full spatula of acid wash sand was added.
- 200ul of pre- heated at 65 0 c SDS was added in the tube - Using sharp pointed pestle, the specimens were crushed till no membrane was physically seen - 400ul of the same preheated SDS was added to make a volume of 600ul - Crushing continued to ensure that the jiggers and flea samples membranes were completely dissolved. - Samples were incubated at 65 0 c for 30mins - 600ul of Chroloform: Isoamylalcohol ratio 24:1 was added into sample tube - The tube was inverted twice to mix. - The solution was centrifuged at 10,000rpm for 10minutes at Room temperature - 500ul of the aqueous upper layer was transferred to a new eppendorf tube taking care not to include any intermediate cell waste layer. - 350 ul of iso propanol (0.7Vol) from (-20 0 c) was added after thawing. - The tubes were centrifuged at 10 000 rpm for 10 minutes at 4 0 c o The pellets formed at the bottom of the tubes - All the supernatant was discarded o The pellet remained at the bottom ready for wash with 70% ethanol - 350 ul of 70% cold ethanol was added to the tube for washing the pellet - The tubes were then centrifuged at 8000rpm for 5mins at room temperatures - The supernatant was discarded. - The Pellet were air dried for 30 Mins - 100 ul of Low salt TE was added to the tube and the pellets dissolved - 2ul of Rnase A was added into the tubes and incubated at 37 0 c for 30 Minutes - 10ul of 3M sodium acetate and 20ul of iso- proponal were added into all tubes o Sodium acetate increase the ionic strength to form the pellets - The tubes were incubated on ice for 10mins o To avoid the Dnase activity - They were then centrifuged at 1000rpm for 10 minutes at 4 0 c - The supernatant was discarded - The formed pellets were washed in 70% ethanol then air dried - 100ul of T.E was added into the tubes for storage
o It solubilize the DNA and to protect it from degradation) PROCEDURE TWO OBJECTIVE: EXTRACTION OF JIGGER AND TUNGA PENETRANS DNA USING CTAB METHOD
- Fall '14