Electroelution+with+Elutrap

Electroelution with Elutrap

Official elutrap documentation

-http://www.whatman.com/ElutrapElectroelutionSystem.aspx#SupportDocumentation -purifies nucleic acids 14 bp to 150 kb

Two membranes are used -BT1 --an inert membrane with a dense matrix through which buffer ions and molecules less than 3-5 kd can pass under the influence of an electric field. --Elutrap-Membranes Whatman 10404090 BT 1 --stored on the "common" 4 C shelf -BT2 --a microporous membrane that acts as a prefilter that prevents agarose and other particulates from entering the purified sample --Elutrap-Membranes Whatman 10404092 BT 2 --stored at room temp in Felicia's drawer

Size of chamber formed by two opposing BT1 membranes: 1.2X10 cm (20 mL capacity)

Protocol -Insert a BT1 membrane between a U-insert and a pressure plate at the front and end of the chamber. --Notched sides of U-inserts should be facing in toward the electroelution chamber. --The higher point on the sloping side of the BT1 membranse should be placed next to the triangular markers on the side of the Elutrap device. The correct orientation of both the BT1 membranes helps maintain the ionic equilibrium of the system and the fluid level in the channel and trap.

-Place a BT2 membrane between a selected pair of U-inserts on the "trap" end. The area between two of the U-inserts can hold from 200-800 uL buffer; the placement of the BT2 membrane will determine the final volume of the trap.

-Place gel slice into sample chamber and add buffer to the entire chamber so that the buffer levels in each compartment are equal. Gel slices should not extend beyond 6 cm. Buffer levels in the Elutrap device should be enough to cover the gel slices or to achieve the final eluate volume desired.

-Note that free space to the side of the electroelution device can be blocked off with a non-conductive material such as a piece of plexiglass or a weighted plastic box to eliminate heat dissipation in the buffer and to minimize power output.

-Apply an electric field strength of approximately 6 V/cm. Overnight elutions should be run at 1/2 the voltage. --Length of medium "100 mL gel" chambers is 10.9 cm. 80% of the voltage power source is applied to the gel. y*0.8/(10.9*cm) = 6 V/cm -> y = 81.8 V --Length of medium "350 mL gel" chambers is 27.5 cm. 80% of the voltage power source is applied to the gel. y*0.8/(27.5*"cm") = 5*"V"/"cm" -> y = 206 V ---Note that it is probably much better to use the "350 mL gel" chambers since the elutrap seems a little too large for the "100 mL gel" chambers.

-After elution is completed reverse the polarity for approximately 20 s to remove any material that may be attached to the BT1 membrane. Remove eluate quickly and reaspirate the fluid a few times to collect the sample material on the surface of the device.

-Remove and discard membranes. Wash the device with distalled water.