5'-end labeling of oligonucleotides - asking for help (Feb/19/2004 )
First question, does any of you buy chemically synthesized oligonucleotides from companies like Qiagen? I just purchased several HPLC-purified DNA oligos from Qiagen. I dephosphorylated some and 5'-end labeled to check purity, but still found several bands. I suspected it is not due to the purity of the oligonucleotides, but due to my experiments, since those bands have similar strength. Have this ever happened to you? Any advice or suggestion would be really appreciated.
Second question, during ethanol precipitation, i found in many protocols that after the supernatant is removed, 70% ethanol is added again and same procedure repeats. what is the purpose for the second addition of ethanol? can this be omitted? should the tube be vortexed or just add the 70% ethanol and recentrifuge?
Sorry for so many questions. i am the only one doing biochemical experiments in my lab. i can't find anyone to ask for help. i am really lucky to find this website.
1. Unless you specifically ordered phosphorylated oligos, they are routinely synthesized without a phosphorylated 5' end and can be kinased directly.
2. When kinasing oligo synthesis products, even when HPLC-purified, you must add a 10-fold excess of label (pmole label:pmole oligo) to avoid conditions which favor labeling the smaller abortive products over the full-length material.
3. If you are doing a film exposure, the linear response is so small (1.2 logs under the best conditions) that you will think you have more short products than are actually there.
4. Ethanol precipitation: the 70% ethanol rinse is required to remove residual salts (and isopropanol if that was used for the precipitation step). Do not omit this step. After centrifuging the ethanol precipitate, carefully remove the supernatant while avoiding the pellet. Then add 1 mL of 70% ethanol, invert the tube a few times to mix, chill and spin for 2 to 3 minutes. Remove the supernatant. Do not overdry the pellet (makes it difficult to resuspend).
Thank you so much!
T4 poly nucleotide kinase is strongly inhibited by amonium ions. I think those are present after synthesis of the oligos. So it might be that an extra wash is done to get rid of most of these incase you want to phosphorylate the oligos using this enzyme.
According to Maniatis, 7 mM (NH4)2SO4 inhibits the forward reaction by 50%.
Oligos are desalted in order to remove the contaminating ammonium ions. Desalting is the minimum level of purification offered by most suppliers. If you ordered HPLC-purified oligos, the presence of contaminating ammonium ions is unlikely.