I digested and dephosphorylated a vector, and digested a PCR amplicon (my insert).
Since I had to use a column to cleanup my vector digest before ligation (since CIP is not fully heat-inactivated), I just decided to cleanup my insert as well.
When I eluted the digested/dephosphorylated vector, I have:
- Conc: 150 ng/uL
- 260/280: 1.8
- 260/230: 1.6
This seems good enough
For the digested insert, though, I have:
- Conc: 90 ng/uL
- 260/280: 1.8
- 260/230: 0.7
Will the low 260/230 inhibit the ligation (T4 ligase) that I plan to do next? Is it worth re-doing the insert digest?
I used a Qiagen kit for the column purification, and I washed it with the wash buffer twice -- maybe this wasn't enough?
The low 260/230 ratio means that you have some contamination by guanidine thiocyanate, which absorbs strongly at 230 nm. However, given that you will dilute the insert in the ligation mixture, the final concentration of guanidine thiocyanate should not affect the ligation significantly.
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