Alice:LabNotes/2010-4-25

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Solexa sequencing library construction (for use with Nimblegen's kit)[edit]

  • sample: CViF (previous steps can be found under labnote 4-22-2010

Washing and Recovery of Captured DNA)[edit]

Step 1. Prepare Sequence Capture Wash Buffers[edit]

  1. 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.
  2. 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]

  1. 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.
  1. Vortex for 20 seconds and label the tube appropriately.
  2. 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]

  1. Allow the Streptavidin Dynabeads to warm to room temperature for 30 minutes prior to use.
  2. Mix the beads thoroughly by vortexing for 1 minute.
  3. 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.
  4. 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.
  5. 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.).
  6. Remove the tube from the DynaMag-2 device and vortex for 10 seconds.
  7. Place the tube back in the DynaMag-2 device to bind the beads. Once clear, remove and discard the liquid.
  8. Repeat Steps 3.5 - 3.7 for a total of 2 washes.
  9. 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.
  10. Aliquot 100μl of resuspended beads into new 0.2ml tubes.
  11. Use the DynaMag-2 device to bind the beads by holding the tube against the magnet. Remove and discard the liquid when clear.
  12. 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]

  1. Transfer the hybridization samples to the Streptavidin Dynabeads prepared in Step 3.12 of this chapter.
  2. Mix thoroughly by pipetting up and down 10 times.
  3. 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]

  1. After the 45-minute incubation, transfer the entire content of each 0.2ml tube to a 1.5ml tube.
  2. Use the magnet from the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear.
  3. Add 100μl of SC Wash Buffer I heated to 47°C. Mix by vortexing for 10 seconds.
  4. Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear.
  5. 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.
  6. Incubate at 47°C for 5 minutes.
  7. Repeat Steps 5.5 - 5.7 for a total of 2 washes with Stringent Wash Buffer heated to 47°C.
  8. Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear.
  9. 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.
  10. Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear.
  11. Add 200μl of room temperature SC Wash Buffer II and mix by vortexing for 1 minute.
  12. Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear.
  13. Add 200μl of room temperature SC Wash Buffer III and mix by vortexing for 30 seconds.
  14. Place the tubes in the DynaMag-2 device to bind the beads. Remove and discard the liquid once clear.
  15. Remove the tubes from the DynaMag-2 device and add 50μl PCR grade water to each tube of bead-bound captured sample.
  16. 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.
  1. Prepare the Post-Capture LM-PCR Master Mix in a 1.5ml tube.
  2. Vortex the bead-bound captured DNA to ensure homogenous mixture of beads.
  3. Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 5 reaction wells.
  4. 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) x17 cycles -> 72C 5min -> hold at 4
  • Qiaquick column purification and elute in 40ul EB
  • PAGE size selection 300-500bp

File:ZhangLab 2 2010-04-26 17hr 15min.jpg

  • PAGE gel quantification:

File:ZhangLab 2 2010-04-27 13hr 37min.jpg

  • CViF:84.67nM