Experiment 7: Preparing a "balanced mix"
Wednesday, June 28, 2000
Location: You will meet in room Z 744 for this week's experiment
(Verfügungsgebäude, 7th floor level).
Teaching Assistant: Your teaching assistant for this experiment is Charles
Information on the experiment.
The title of today's experiment is "preparing a balanced mix". It turns out that the "balanced mix" is a jargon term created in these laboratories. It is used to describe a library of compounds whose concentration is adjusted such that all of the compounds can be followed through a spectrometrically monitored selection experiment (SMOSE). Usually, this means an equimolar amount of all library components. Only in some cases, when one of the library members has a particularly low desorption/ionization efficiency, or if a selection experiment is done at the very detection limit, so that one of the compounds would otherwise fall out of the detection range, is a non equimolar ratio of library components used. The goal of today's experiment is to prepare an at least roughly equimolar mixture of your 5'-acylated oligonucleotides and to ensure that all library members can be detected via quantitative MALDI-TOF mass spectrometry.
Since your last experiment, Jason Mayo and Andriy Mokhir have prepared stocks of pre-purified oligonucleotides for you. In some cases, this involved re-synthesizing your compounds and pre-purifying mixtures again via HPLC. Further, a UV spectrum of an aliquot of each pre-purified compound was acquired to allow for quantifying each compound. You will be provided with the UV reading at 260 nm from the spectrum, together with the MALDI spectrum of the pre-purified compound.
Experience shows that the more heavily modified oligonucleotides, particularly if the appended non nucleic acid residue is charged or amphiphilic, give less intense MALDI peaks than their unmodified counterparts. So, you should not be surprised, if in what appears to be an equimolar ratio of your oligonucleotide hybrids and the acetylated control compound, the peak intensities are not 1:1:1:1:1.
Detailed recommendations for the experiment follow below.
Preparing a balanced mix. You will obtain stock solutions
of the pre-purified compounds of a total volume of 10 µL. Based
on the MALDI spectrum that you will obtain, you will be able to estimate
the purity of each compound (remember that shorter sequences fly better
than longer ones, so smaller molecules are usually overrepresented, giving
you a lower limit of purity, if you estimate by peak height or integration).
With the UV reading of a diluted aliquot of your stock solutions, you can
now calculate the concentration of every of your compounds. Please
take an aliquot that contains 15 pmol of your compound from each solution
and pool these aliquots into one Eppendorf cup. For many of your
solutions, you will have to do a 1:10 dilution first (take out 1 µL
and add 9 µL of water) before you can accurately pipette a volume
containing 15 pmol of your compound. Remember that 0.5 µL is
the smallest volume that can be somewhat accurately pipetted with the smallest
pipetmen that are available. To this mix of 4 x 15 pmols, one aliquot
of 1 µL from the stock solution of the control oligonucleotide (Ac-C*GGTTGAC,
where C* is a 5'-amino-5'-deoxycytidine residue) should be added.
This stock solution will contain approximately 14-15 pmol of Ac-C*GGTTGAC
per µL and will be provided by your teaching assistants. The
result should be an equimolar mixture of your four modified oligonucleotides
and the control oligomer that is acetylated.
Now, fill up the mixture to a defined volume, ideally 10 µL. Of this, 1 µL should be mixed with a matrix mixture for MALDI mass spectrometry, consisting of 2.5 µL trihydroxyacetophenone solution in ethanol and 1.5 µL of diammoniumcitrate solution in water. Again, these solutions will be provided by your teaching assistants. Mix carefully and apply 1 µL of the resulting mixture to a spot on a MALDI target. Please document every step, and enter your name and compound mix identifiers on the log page for the MALDI target. Your teaching assistant will now acquire a MALDI spectrum of your mixture. Take this spectrum, measure the relative peak intensities of your compounds (also check that they appear at the calculated mass ± 2 mass units). If the peak intensity for the components differs by more than a factor of 2, adjust the concentration of the more poorly detectable compounds in the mixture and have a new spectrum acquired, until all compounds can be detected within this intensity range of 2-fold relative intensity.
A Note at the End. By now, a very substantial amount of work has been invested into preparing your samples. Please be as careful as possible when handling them and when planning each step of your procedure. Please double check each calculation, so that the unfortunate loss of material that occurred in an early experiment of this course does not happen again.