QUESTION: What are the relative extinction coefficients of 5'-Fluorescein, Hex and Tet etc.. at 260 nm and their Lambda max?
QUESTION: Why does MALDI analysis of my oligos that contain one or more Fluorescein-dTs give an incorrect mass even though they give only a single, fluorescent band on a PAGE gel?
RESPONSE:It's an artifact of the MALDI analysis. The lasers used to ablate the MALDI matrix are generally between 308 and 355 nm, with the most frequently used laser line at 337 nm. When a laser hits a strong absorbance band of a molecule, often photochemistry starts to occur - which often leads to cleavage reactions. The Fluorescein-dT has strong UV absorbance in this region which appears to lead to a 135 m/z fragment is being blown off the molecule in a rather consistent manner. For instance, your oligos D1 and D2, which have 5 and 3 Flu-dTs respectively, the observed mass difference is -676 Da and -406 Da, which nicely fits the loss of a 135 mw fragment: 5 x 135 (675) and 3 x 135 (405). We haven't seen any papers that identify this 135 m/z fragment - but it's clear to me that that is what's occurring. Changing matrix matrix used may help. The matrix 2,4,6-trihydroxyacetophenone has been used successfully to analyze a fluorescein-labeled oligos, though the safest bet is to use Electrospray MS rather than MALDI for mass spec analysis.
QUESTION: What are the relative extinction coefficients of various dyes?
QUESTION: Does AMA or methylamine cause any degradation to fluorescein or fluorescein-type dyes such as FAM or FITC?
RESPONSE:Response: While AMA (Ammonium hydroxide/40% Methylamine 1:1 v/v) is considered compatible with fluorescein, the use of methylamine when deprotecting a Fluorescein-labeled oligo does lead to a small amount of degradation, which is characterized by a the appearance of a late-eluting peak by RP HPLC that shows no visible fluorescein absorbance. With standard deprotection conditions (AMA 10 minutes at 65 C) the amount of degradation is approximately 5%. The impurity is not detected with AMA at RT for 2 hours.
If you cannot find the answer to your problem below then please
contact us or telephone 01954 210 200