I guess the molecular mass of your DNA (90mer) you are using for ligation is lower than the detection level of (even sensitive) SYBR gold. Perhaps you can order a oligo to make more copies of your 90mers from the ligated product(by PCR). Of course, they may amplify the unligated DNA as well, but you may know how to separate them cleverly. If I were you, I will simply order this oligo (at least HPLC purified or higher; a 80mer cost me around 40€) from a company and do my assays using it :)

Hi Ananda Ayyappan Jaguva Vasudevan - I did order the 90mer oligo to use as a control and tried different dilutions of that. I do like your suggestion of PCR amplification but my limiting factor will be the adduct I am adding to one of the oligo's.

The phosphorylation with radioactivity or another label could maybe help, or a higher concentration of Sybr Gold.

Have you ever tried EtBr instead it is more powerful than other although the more toxic

Hi Marion,

Thought about labeling but we are unable to do radioactivity in our lab. Ordering the oligos already labeled with TMR is pricey.

Shahram - SYBR gold is supposed to be 25X more sensitive than EtBr. Initially I tried EtBr.

Hi Rochelle, did you already thought about doing it blind. You could estimate the region where it should be on the gel, isolate it and reamplify.

I can try Marion. Thanks!

Is the purpose of ligation to clone the "ligated oligo"?If it is so, then I would suggest multiple insert ligation with a vector in two steps i) ligate oligo, add equal volume chloroform, centrifuge at high speed for 3 min. ii) carefully use the supernatant for ligation with vector in the subsequent ligation step. proceed to same purification method as above and ELECTROPORATE.

Hope It helps.

Hi Rochelle, I have routinely ordered upto 200nt "Ultramer" oligos, gel-purified, from a company called IDT(Integrated DNA Technologies) & they've consistently performed well. They have the option of a 4nmole or 20nmole synthesis + PAGE purification & may save you a lot of time. Ron Pithawalla

If you want a vector with your 90mer oligo, why don't you synthesis directly inside a vector by a service company (eurogentec, sigma, euorfins etc...). For only 90mer it might be not so expensive.

Hello all,

Actually Rochelle needs her oligo to be labelled with TMR (tetramethylrhodamine-5-iodacetamide?) and so she wants to ligate a fragment having TMR and a fragment without(am I right?). For synthesis, a 90mer with TMR may be too expensive.

Hi -

I really appreciate all the replies and suggestions. What a great community! I will not be cloning my oligo or using it as a vector. It is for a sensing project. My limiting factor is the DNA adduct that I need to add to one of the fragmens prior to ligation. We thougth about labeling the ends with TMR but that is cost prohibitive. At this point we are going for th with the experiment since our yeild (via qPCR) was around ~40%.

I appreciate the help!

Cheers,

Rochelle

Gel-red is one powefull and sensitive alternative. You may want to try it out.

Often times with RNA seq we are ligating sequencing adapters onto to very small amounts of cDNA.so we just cut the etbr agarose gel blindly according to the ladder, qiagen gel extraction kit, and then pcr amplify the product to test for size distribution. It's always there, despite not being able to see it.

I dont know if the addition of a couple of restriction endonuclease sites to the ends of your 90 bp oligo is OK with your downstream experiments (or it may already have some sites). If it is OK then I would clone it to any plasmid, so you'll have an unlimited source of your oligo. Than digest plasmid, purify oligo and label it with any chemicals