concentration of TruSeq adapter stocks?

[jlove]

Does anybody know what the concentration of the TruSeq adapters is? I am currently making my own set and want to match it up to whatever Illumina is doing. They have told me that the old adapters are 15 uM but that the TruSeq adapter concentration is proprietary. Does anybody know any details?

Thanks in advance

[ETHANol]

Thought I'd revive this thread. Does anybody know the concentration of the TruSeq adapters stocks, from either the DNA or RNA sample prep kits?

From another thread, I've found that the DNA adapters are 60x the concentration of the RNA adapters.

Illumina confirms they are both methylated (5MeC).

I don't have the kits so I can't spec them at 260nm. I just want to know how much I get with the kits.

[jlove]

I'll follow up to my own question:
I made my adapters at a couple of different concentrations ranging from 25 uM to 8 uM (each) and compared them to the Illumina ones by denaturing them and running them out on the smRNA assay on the bioA. I ultimately landed on something right around 15 uM (each) being the closest, so that's what I made my stocks out to be. For what it's worth, I did not methylate my adapters. It's really good to know that the RNA ones are about 60x the DNA ones! Do you have a link to that thread?

[ETHANol]

http://seqanswers.com/forums/showthread.php?t=13911

Just to be clear the RNA adapters are 60 fold more dilute then the DNA adapters.

15 uM is that for the RNA or DNA adapter stocks?

[jlove]

15 uM each is what I found to be equivalent to the *DNA* sample prep kit.

[ETHANol]

Thanks!!

I was hoping to use the adapters from the RNA kit for RRBS-seq and replace them with unmethylated oligos (methylated oligos are expensive) but it looks like they are too dilute.

[pmiguel]

Illumina confirms they are both methylated (5MeC).

Why use 5MeC?

--
Phillip

[jlove]

It's for methylation studies

[protist]

It states in the original Illumina Patent (see page 50 of doc) that the adapters are at a concentration of 15 uM. Of course that was pre-TruSeq adapters..but for our home-brew stocks the masters are kept at 15 uM and diluted when necessary.

[pmiguel]

Insert:Adapter ratios TruSeq DNA

Years ago I took course in Molecular Genetics from Irwin Tessman here at Purdue. One of the non-biological central tenants of the course was that we were not to use calculators on exams. One basis for this practice is that using the calculator allows one to arrive at answers that given a bit of thought would be obviously incorrect.

Along those same lines I think it is productive to think about protocols as to what is being accomplished in each step and what problems each step causes for the next step. But even more centrally one wants to ask:

What and how many objects (molecules) are being modified in a given step?

As I have mentioned before, I use the rule of thumb:

1 ug of DNA is 1 trillion 1 kbp fragments. (Actually only about 93% of this, but close, enough for most purposes.)

The TruSeq DNA prep kit starts with 1 ug of DNA as measured by a double-strand specific method (eg PicoGreen fluorimetry). If we naively presume that 50% of the DNA makes it through AMPure purification after sonication/end repair that would leave us with just under 500 billion 1000 bp molecules. But sonication has broken the molecules into 500 bp on average, at most, not 1000 bp. So double that back to 1 trillion insert molecules.

Up thread it is estimated that TruSeq adapters have a concentration of 15 uM. That brings me to my second rule of thumb:

1 ul of a 1 uM solution contains 1 trillion molecules. (Actually only about 60% of this, but we can refine answers after an initial calculation, if necessary.)

So 15 uM would be about 15 trillion adapter molecules per ul. Let's multiply by .6 (60%) to refine the estimate to 9 trillion/ul. 2.5 ul of adapter is used in each ligation. So a total of 22.5 trillion adapters are being added to the ligation.

That puts the Insert:Adapter ratio around 1:20. That makes the insert end:adapter ratio about 1:10.

Of course the actual amount of insert going into the reaction is going to be quite variable.

--
Phillip