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==Washing and Recovery of Captured DNA (done on 4/5/2010)== ===Step 1. Prepare Sequence Capture Wash Buffers=== # Dilute 10X SC Wash Buffers (I, II, and III) and 2X Stringent Wash Buffer to create 1X working solutions. Store working solutions at room temperature for up to 2 weeks. # Preheat the following wash buffers: 20ml of Stringent Wash Buffer heated to 47°C in a water bath and 5ml of SC Wash Buffer I heated to 47°C in a water bath. ===Step 2. Prepare Streptavidin Dynabead Binding and Wash Buffer=== #Prepare the Streptavidin Dynabead Binding and Wash Buffer in either a 15ml or 50ml conical tube: for every 4 capture reaction, the following components are needed: 1M Trizma hydrochloride: 25ul 0.5M EDTA: 5ul 5M NaCl: 1000ul PCR grade water: 1470ul total: 2.5ml Volume adjusted for pipetting variance. Store wash buffer at room temperature for up to 2 months. #Vortex for 20 seconds and label the tube appropriately. #Store the Streptavidin Dynabead Binding and Wash Buffer at room temperature while proceeding immediately to “Step 3, Prepare the Streptavidin Dynabeads.” ===Step 3. Prepare the Streptavidin Dynabeads=== #Allow the Streptavidin Dynabeads to warm to room temperature for 30 minutes prior to use. #Mix the beads thoroughly by vortexing for 1 minute. #Aliquot 100μl of beads for each capture into a single 1.5ml tube (i.e. for 1 capture use 100μl beads and for 4 captures use 400μl beads, etc.). Enough beads for 6 captures can be prepared in a single tube. #Place the tube in a DynaMag-2 device. When the liquid becomes clear (should take less than 5 minutes), remove and discard the liquid being careful to leave all of the beads in the tube. Any remaining traces of liquid will be removed with subsequent wash steps. #While the tube is in the DynaMag-2 device, add twice the initial volume of beads of Streptavidin Dynabead Binding and Wash Buffer (i.e. for 1 capture use 200μl of buffer and for 4 captures use 800μl buffer, etc.). #Remove the tube from the DynaMag-2 device and vortex for 10 seconds. #Place the tube back in the DynaMag-2 device to bind the beads. Once clear, remove and discard the liquid. #Repeat Steps 3.5 - 3.7 for a total of 2 washes. #After removing the buffer following the second wash, resuspend by vortexing the beads in 1x the original volume using the Streptavidin Dynabead Binding and Wash Buffer (i.e. for 1 capture use 100μl buffer and for 4 captures use 400μl buffer, etc.) that was prepared in Step 2.3. #Aliquot 100μl of resuspended beads into new 0.2ml tubes. #Use the DynaMag-2 device to bind the beads by holding the tube against the magnet. Remove and discard the liquid when clear. #The Streptavidin Dynabeads are now ready to bind the captured DNA. Proceed immediately to “Step 4, Bind DNA to the Streptavidin Dynabeads.” ===Step 4. Bind DNA to the Streptavidin Dynabeads=== #Transfer the hybridization samples to the Streptavidin Dynabeads prepared in Step 3.12 of this chapter. #Mix thoroughly by pipetting up and down 10 times. #Bind the captured sample to the beads by placing the tubes containing the beads and DNA in a thermocycler set to 47°C for 45 minutes. Mix the samples by vortexing for 3 seconds at 15 minute intervals to ensure that the beads remain in suspension. It is helpful to have a vortex mixer located close to the thermocycler for this step. ===Step 5. Wash the Streptavidin Dynabeads Plus Bound DNA=== #After the 45-minute incubation, transfer the entire content of each 0.2ml tube to a 1.5ml tube. #Use the magnet from the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear. #Add 100μl of SC Wash Buffer I heated to 47°C. Mix by vortexing for 10 seconds. #Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear. #Remove the tubes from the DynaMag-2 device and add 200μl of Stringent Wash Buffer heated to 47°C. Pipette up and down 10 times to mix. Work quickly so that the temperature does not drop much below 47°C. #Incubate at 47°C for 5 minutes. #Repeat Steps 5.5 - 5.7 for a total of 2 washes with Stringent Wash Buffer heated to 47°C. #Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear. #Add 200μl of room temperature SC Wash Buffer I and mix by vortexing for 2 minutes. If liquid has collected in the tube’s cap, tap the tube gently to collect the liquid into the tube’s bottom before continuing to the next step. #Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear. #Add 200μl of room temperature SC Wash Buffer II and mix by vortexing for 1 minute. #Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear. #Add 200μl of room temperature SC Wash Buffer III and mix by vortexing for 30 seconds. #Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear. #Remove the tubes from the DynaMag-2 device and add 50μl PCR grade water to each tube of bead-bound captured sample. #Store the beads plus captured samples at -15°C to -25°C or proceed to Chapter 7, Captured DNA Amplification Using LM-PCR. *Note: There is no need to elute DNA off the beads. The beads plus captured DNA will be used as template in the LM-PCR.
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