CTR:Notebook/PIRE/2014/11/21

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RAD P1 Adapter Ordering

Adapter Sequences (sequences from Mike Miller's files)
Ordered in plate format from IDT (1 plate of top oligos, 1 plate of bottom oligos)
Parameters:

  • Scale: 100 nmole
  • Purification: Standard desalting
  • Ship option: Wet
  • Amount per well: Full yield
  • Concentration: 100 μM
  • Dilutant: IDTE Buffer pH 8.0

RAD P1 Adapter Annealing and Dilution

Based on instructions from Mike Miller's RAD adapter information file.

1. Dilute oligo plates from 100μM to 10μM

  • To make 20μL plate, take 2μL of 100μM stock and dilute in 18μL dH2O.

2.Make adapter mix plate (20μL)

  • For each well:
    • To get 10mM Tris, need 0.2μL of 1M stock Tris
    • To get 50mM NaCl, need 0.2μL of 5M stock NaCl
    • To get 2μM each oligo, need 4μL of 10μM each oligo
  • For 96 well plate, make a 110x mix of 22μL of 1M Tris, 22μL of 5M NaCl, and 1276μL of dH2O into a microtube, then transfer to reservoir. Pipette 12μL of mix into each well, then add 4μL of top and bottom oligo to each well for a total of 20μL.

3. Anneal adapters

  • 98°C for 2 minutes
  • Ramp: 98°C to 10°C for 20 minutes

4. Make dilution plate (195μL of 10mM Tris, 50mM NaCl)

  • For each well, need:
    • 1.95μL of 1M Tris
    • 1.95μL of 5M NaCl
    • 191.1μL of dH2O
  • For 96 well plate, make 110x mix of 214.5μL of 1M Tris, 214.5μL of 5M NaCl, and 21.021mL dH2O into falcon tube, then transfer to reservoir. Pipette 195μL of mix into each well.
  • Add 5μL of annealed adapter to the dilution plate. The adapter concentration is now 50nM.

5. Make final adapter plates (10nM)

  • For each plate, take 2μL of 50nM stock adapter and dilute in 8μL of dH2O.