Use of degenerate bases

Spoke with IDT about some of their recommendations related to incorporation of degenerate bases in oligos.

Machine Mix vs Hand Mix option

  • Machine Mix:
    1. Bases are mixed at equal molar concentrations.
    2. Each base has a different coupling efficiency which leads to different incorporation rates.
      • Automated message states that this is a difference in delivery rates, but that appears to be somewhat misleading.
    3. Results in the following incorporation ratio for a single "N":
      • 30:26:24:20 T>G>C>A
    4. Ratio is per base, and a string of N's does not change the ratio.
    5. No additional charge.
  • Hand Mix:
    1. Can be used to correct the machine mix ratio.
    2. Percents entered are the incorporation percents, not the mixture percents.
      • ie: entering 25:25:25:25 would result in them decreasing the amount of T and increasing the amount of A to achieve 25:25:25:25 final diversity.
    3. Claim their formula is accurate to within less than 1%.
    4. Additional cost required.

PAGE and HPLC problems

  1. Each base (A,C,T,G) has a slightly different migration rate than each other.
  2. IDT uses a very conservative selection to ensure that all product is full length.
  3. Results in loss of diversity.
  4. Works against hand mixing.
  5. Suggested that can try standard desalting and doing our own less conservative PAGE purification to eliminate truncated product. Warning that this can still lead to elimination of some diversity.

-- Main.DanielDeatherage - 22 Jan 2013


This topic: Lab > WebHome > ProtocolList > ProtocolsDegenerateBases
Topic revision: r1 - 22 Jan 2013 - 17:25:56 - Main.DanielDeatherage