Matt Bowser – order 4869 – Used Omega’s Mag-Bind Plant DNA DS kit optimized for Kingfisher
1.) Insects were removed from the propylene glycol antifreeze and placed into 15ml centrifuge tube.
2.) Using 100% Ethanol, insects were washed twice with approximately 2-3ml in order to remove residual propylene glycol. Using fresh 100% Ethanol, the insects were covered and stored at room temperature for 1 week.
3.) After 1 week, the ethanol was removed and discarded.
4.) Using Phosphate Buffered Saline (PBS), the insects were washed with approximately 2-3ml to remove residual ethanol. Covering the insects with fresh PBS, they were stored at room temperature for a weekend.
5.) After sitting in PBS over the weekend, the PBS was removed and discarded.
6.) Using an Omni Tissue Homogenizer, the insects were thoroughly homogenized.
7.) Using a small spatula, a pea size amount of the insect homogenate was loaded into a 2ml deep well plate.
8.) To each well, 700ul of CSPL buffer and 20ul of Proteinase K was added.
9.) The plate was sealed using a thick Adhesive seal and vortexed.
10.) After a quick spin, the plate was incubated at 56°C for 1 hour.
11.) Centrifuge plate at 4500g for 10 minutes
12.) Transfer 475ul of lysate to a new deep well plate.
13.) To each well, 475ul of RBB buffer and 20ul of MagBind HDQ particles was added.
14.) Make CSPW1 wash plate – each well contains 500ul wash buffer.
15.) Make CSPW2 wash plate – each well contains 500ul wash buffer.
16.) Make SPM wash plates – each well contains 500ul wash buffer.
17.) Make water rinse plate – each well contains 500ul of DNase/RNase, Protease and Endotoxin free Molecular Grade Water
18.) Make elution plate – each well contains 50ul of elution buffer.
19.) Place plates on the Kingfisher Machine using Omega M1130_50 program.
 a. Binding step – beads are mixed for 1 minute at fast speed, 1 minute at bottom mix, then 1 minute at half mix, repeating this mixing for a total of 3 times.
 b. CSPW1 Wash 1 – beads are mixed for 2 minutes at fast speed
 c. CSPW2 Wash 2 – beads are mixed for 1 minute at fast speed
 d. SPM Wash 3 – beads are mixed for 1 minute at fast speed
 e. SPM Wash 4 – beads are mixed for 1 minute at fast speed
 f. Water Dip – beads are dipped into water for 5 seconds
 g. Elution – beads are mixed for 2 minutes at slow speed, then bottom mixed for 1 minute in the elution buffer with heat at 65°C. It is repeated following the same mixing pattern. The beads are removed from the elution buffer and dropped into wash 4 plate to be discarded.