Oligonucleotide dilution

[Erin Wu]

Hi,

    I have a question, you recommend  diluting oligos using 1X TE + 0.05% Tween 20, but our lab always use RNase free water. Does it affect downstream reactin?

[Joe Foley]

It's probably fine for the short term and there's no significant effect on the reactions, but for long-term storage it's preferable to store DNA in a buffered solution with a pH around 8 at 25 °C. Pure water will tend to have a pH below 7 as it reacts with atmospheric carbon dioxide to form carbonic acid. More information: https://www.idtdna.com/pages/education/decoded/article/storing-oligos-7-things-you-should-know

EDTA is less essential, since it protects from degradation catalyzed by divalent cations (the same principle used in this protocol to fragment RNA), but you probably don't have those in your tubes. Tween 20 is only there to reduce surface tension and increase pipetting accuracy, which is not a big deal for this protocol - that's normally used for qPCR. But I find it easier to make hundreds of milliliters of the best possible storage solution and use that for everything rather than maintain a variety of different diluents for different purposes.

At any rate, the indexed PCR primers in particular are a big investment and it takes a long time to use them up, so I think if you spend hundreds of dollars on deluxe custom oligos you can afford twenty bucks for TE to ensure they're stored safely.

On 8/12/20 11:48 PM, Erin Wu wrote:

Hi,

    I have a question, you recommend  diluting oligos using 1X TE + 0.05% Tween 20, but our lab always use RNase free water. Does it affect downstream reactin?

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[Erin Wu]

OK, thanks for your perfect explanation sincerely! But I have some trouble creating a single libray with standard Smart-3SEQ method. I input 9ng & 0.225ng total RNA, but I alway get a unwanted libray by Agilent 2100 detection. There are seemly too much primer-dimer. I attach my  result, could you give me some suggestion?

[Joe Foley]

The 9 ng library looks like it's roughly the right shape but the yield might be a little low (hard to tell without the numbers, but generally you want more than 1 nM). According to the formula in the protocol, you'd expect the best results from 15 to 16 PCR cycles for 9 ng of high-quality human RNA, though you may need to recalibrate the formula for your own samples.

225 pg is too little to make a good single library with this protocol; the threshold is about 1 ng. If you want to use 225 pg you'll have to use the pre-SPRI pooling option to combine several libraries.

To view this discussion on the web visit https://groups.google.com/d/msgid/smart-3seq/d5c6f642-dbef-4e5a-b1b4-b59600cb2bcfn%40googlegroups.com.

[Erin Wu]

Ok, I see. Thanks for your help！I will try another tests accoding to your suggestion.

[Erin Wu]

Hello,        

Excuse me! I have another question. You said that the low input may be responsible for low yied for an individule library, and your protocol is available for single cell. So if I want to make a library starting from one  single cell, I must combine at least how many cells to get a desired yied?

[Joe Foley]

It depends on the amount of (available) RNA in your cells; even within a single metazoan organism this can vary widely from one cell type to another. If you assume 10 pg per cell, the formula estimates you'll need about 50 single-cell libraries to keep it down to 20 PCR cycles. Single-cell libraries have low information content so you might want more than that just for data quality. First you might test making unpooled libraries from known, higher numbers of cells (e.g. about 50, 100, 200, 400; this is a rough estimate so they don't have to be precisely counted or pure) then add a couple of extra PCR cycles to be safe.

To view this discussion on the web visit https://groups.google.com/d/msgid/smart-3seq/71615258-f654-4ff9-991b-b054ee1ee100n%40googlegroups.com.

[Erin Wu]

Ok, I see. Sincerely thanks for your reply!