Titration of AmPure XP Beads for Removal of Fragments < 400bp
Label 11x1.5 ml tubes 40 - 45 - 50 - 55 etc .
to 90 ( = ratio of beads to DNA ) Mix 48μl NEB 100bp ladder with 1152μl Molecular Biology Water .
Pipet 100μl of mixture into each of the 11 tubes .
Vortex bottle of AmPure XP beads and dispense 800μl into fresh tube .
Using same pipettor used for step # 3 , pipet volume of beads into each tube ( i . e . , 40μl into tube labled 40 , 45μl into tube labed 45 , etc . )
Close lids , vortex tubes and incubate 5 minutes at room temperature .
Place tubes in MPC rack ; wait 5 minutes for beads to adhere to magnet side of tubes .
Pipet off supers and discard them .
Wash the beads 2x with 500μl each 70 % ethanol with 30 second incubation .
Remove all supers from tubes .
Place tubes ( with caps open ) into 37°C heat block to dry beads ( evaporate residual ethanol ) .
Add 10μl Qiagen EB to each tube , close lids .
Vortex to resuspend beads .
Place tubes back into MPC rack and let beads adhere to magnet sides of tubes .
Pipet off supers ( containing eluted DNA ) into fresh tubes .
Run samples in 2 % agarose gel ( 1x TAE ) using 5μl DNA and 1μl 6x gel loading buffer .
Photograph results and determine what ratio of beads to DNA to use to exclude lower range of DNA .
