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==<span style="color:blue">Date of experiment 01/20/2015</span>== ==== IMPORTANT NOTE ==== * It is extremely important to maintain the water bath at 47C. I just have two samples, so I used thermomixer and prepare all washing buffer in 1.5mL or 2mL tube * Equilibrate buffer at 47C for ''at least 2h before washing the captured Multiplex DNA sample. This should mean '''Stringent Wash Buffer and Wash Buffer I''' as they were used at 47C. <br> === Step 1. Preparing Sequence Capture and Bead Wash Buffer === {| {{table}}border = 1 | align="center" style="width:220px;background:#f0f0f0;"|'''Concentrated Buffer''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume/sample (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 2.1X''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O for 2.1X''' |- | 10X Stringent Wash Buffer (vial 4)||40||360||400||84||756 |- | 10X Wash Buffer I (vial 1)||30||270||300||63||567 |- | 10X Wash Buffer II (vial 2)||20||180||200||42||378 |- | 10X Wash Buffer III (vial 3)||20||180||200||42||378 |- | 2.5X Bead Wash Buffer (vial 7)||200||300||500||420||630 |} # Dilute 10X Wash Buffer (I, II, III and Stringent) and 2.5X Bead Wash Buffer to 1X working solutions as table above. ## Warm 1X Wash Buffer I and 1X Stringent Wash Buffer at 47C on thermocycler at least 47C as describe above. I have a very short time to prepare, so I incubated only ~1h. * NOTE: All of Stringent Wash Buffer is used at 47C. Wash Buffer I will be used at 47C and RT (100ul at 47C and 200 at RT per rxn). I May need to aliquot accordingly. <br> === Step 2. Preparing the Capture Beads === * Step below for 2 captures # Allow the capture beads to warm to RT for 30min prior to use. # Mix the beads thoroughly by vortexing to 15s. # Aliquot 100ul of beads for each capture into a 1.5mL tube. For 2 captures, aliquot 200ul. of beads. # Place the tube with beads on magnet device for 1.5mL tube for 3-5min until the liquid is clear. Remove and discard the liquid. # Add 400ul of Bead Wash Buffer (200ul/sample) # Vortex the beads for 10s # Place the tube back to the magnet. Remove the liquid once the liquid is clear. # Repeat step 2.5-2.7 for a total 2 washes # Resuspend the bead with 200ul Bead Wash Buffer and vortex (100ul/sample) # Aliquot 100ul of resuspended beads into new lo-bind 0.2mL tube. # Place the tube on the magnet. Remove the clear liquid. # The capture beads are now READY to bind to captured DNA. '''PROCEED IMMEDIATELY TO STEP 3. BINDING DNA TO THE CAPTURE BEADS''' <br> === Step 3. Binding DNA to the Capture Beads === # Transfer the hybridization samples to the capture beads prepare in step 2.11 # Mix thoroughly by pipetting up and down 10X. NOTE: I have carried a small volume of mineral oil with the hybridization sample as I wanted to transfer as much as possible of the hybridization sample. # Place the tube containing the beads and DNA on a thermocycler set at 47C for 45min (heat lid maintained at 57C). I just keep thermocycler after after hybridization on. ## Mix the samples by vortexing 3s every 15min to ensure that hte beads remain in suspension. Should have vortex close to thermocycler. <br> === Step 4. Washing the Capture Beads Plus Bound DNA === # After 45-min incubation, add 100ul of 1X Wash Buffer I heated to 47C to the 15ul capture beads plus bound DNA. I added while the tube maintained on thermocycler at 47C. # Mix by vortexing 10X # Transfer the entire contents to Lo-bind 1.5mL tube # Place the tube in the magnet to bind the beads. Remove and discard spnt # Remove the tube from the magnet and add 200ul of 1X Stringent Wash Buffer heated to 47C. Pipette up and down 10X. WORK QUICKLY so that the temperature does not drop down much below 47C. ## I place the tube to thermomixter next to Stringent Wash Buffer and add the buffer. # Incubate at 47C for 5min # Repeat step 4.4 - 4.6 for a total 2 washes using 1X stringent Wash Buffer heated to 47C # Place the tube back to the magnet and remove spnt # Add 200ul of RT 1X Wash Buffer I -> mix by vortexing for '''2min''' # Place the tube back to the magnet and remove spnt # Add 200ul of RT 1X Wash Buffer II -> mix by vortexing for '''1min''' # Place the tube back to the magnet and remove spnt # Add 200ul of RT 1X Wash Buffer III -> mix by vortexing for '''30s''' # Place the tube back to the magnet and remove spnt # Remove the tube from the magnet and add 50ul H2O. Mix by vortexing. # Continue to Amplification Step or store the beads plus captured DNA at -15 to -20C. # I stored the captured DNA in -20C '''THERE IS NO NEED TO ELUTE DNA FROM THE BEADS'''<br> <br>
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