Alice:LabNotes/2010-4-2
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Prepare samples for Solexa sequencing (for use with Nimblegen's kit)[edit]
- previous steps can be found under labnote 3-22-2010
- four samples will be processed:
- nanodrop result (40ul total for each):
- 139TD: 187ng/ul (13ul needed)
- 121DB: 201.3ng/ul (13ul needed)
- 56JW: 56.7ng/ul (40ul needed)
- 56WW: 167.9ng/ul (15ul needed)
Hybridization preparation and setup[edit]
- Add 100μl of 1mg/ml COT DNA and 1μg of amplified sample library to a new 1.5ml tube.
- Add 10μl of each 100μM PE-HE1 and PE-HE2 Oligos to the amplified sample library plus COT DNA. (PE-HE1=Solexa_up, PE-HE2=Solexa_lo)
- Close the tube’s lid and make a hole in the top of the tube’s cap with an 18 - 20 gauge or smaller needle. (Note: The hole in the top of the tube’s cap is a precaution to suppress contamination in the SpeedVac.)
- Dry the amplified sample library/COT DNA/PE-HE Oligos in a SpeedVac on high heat (60°C). (Note: This step may take 30 minutes or longer. To minimize drying time, dry COT DNA ahead of time and then add the amplified sample library and PE- HE Oligos. Denaturation of the DNA with high heat is not problematic after linker ligation because the hybridization utilizes single-stranded DNA.)
- To each dried-down amplified sample library/COT DNA/PE-HE Oligos, add 7.5μl of 2X SC Hybridization Buffer and 3μl of SC Hybridization Component A
- Cover the hole in the tube’s cap with a sticker or small piece of laboratory tape. Vortex the amplified sample library/COT DNA/PE-HE Oligos plus Hybridization Cocktail (2X SC Hybridization Buffer + SC Hybridization Component A) for 10 seconds and centrifuge at maximum speed for 10 seconds.
- Place the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail in a 95°C heat block for 10 minutes to denature the DNA.
- Centrifuge the amplified sample library/COT DNA/PE-HE/Hybridization Cocktail at maximum speed for 10 seconds at room temperature.
- Transfer the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail to the 4.5μl aliquot of Exome Library in a 0.2ml PCR tube prepared in Chapter 2 (entire volume can also be transferred to a 96-well PCR plate). Vortex for 3 seconds and centrifuge at maximum speed for 10 seconds.
- Incubate in a thermocycler (with heated lid turned on) at 47°C for 72 hours.
Washing and Recovery of Captured DNA (done on 4/5/2010)[edit]
Step 1. Prepare Sequence Capture Wash Buffers[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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.
Captured DNA Amplification Using LM-PCR[edit]
- A total of 5 reactions are performed per sample, and subsequently combined, to minimize PCR bias.
- Note: The Post-Capture LM-PCR Master Mix and the individual PCR tubes must be prepared on ice.
- Prepare the Post-Capture LM-PCR Master Mix in a 1.5ml tube.
- Vortex the bead-bound captured DNA to ensure homogenous mixture of beads.
- Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 5 reaction wells.
- Aliquot 10μl of bead-bound captured DNA as template into 5 PCR tubes/wells.
The amount of each reagent needed for each reaction is listed below: Phusion High-Fidelity PCR Master Mix (2x) 50ul PCR grade water 36ul Syb_FP5, 100 μM (Final Conc.: 2μM) 2ul Syb_RP7, 100 μM (Final Conc.: 2μM) 2ul Total 100ul 98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C TBE gel image result:
- Qiaquick column purification and elute in 40ul EB