META TOPICPARENT |
name="ProtocolList" |
# 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:
- Bases are mixed at equal molar concentrations.
- 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.
- Results in the following incorporation ratio for a single "N":
- Ratio is per base, and a string of N's does not change the ratio.
- No additional charge.
- Hand Mix:
- Can be used to correct the machine mix ratio.
- 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.
- Claim their formula is accurate to within less than 1%.
- Additional cost required.
## PAGE and HPLC problems
- Each base (A,C,T,G) has a slightly different migration rate than each other.
- IDT uses a very conservative selection to ensure that all product is full length.
- Results in loss of diversity.
- Works against hand mixing.
- 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 |