SYBR Green I - (Jan/25/2006 )

Hi
has anyone had any issues with Sigma's SYBR Green I Product No S 9430?? we have been previously using a vial (about 4 years old) and had no problems using it at 1x concentration but with the new vial at 1x it inhibits the PCR and we need to use it at 0.1x. has anyone else found this?? we have tried it on three new batches of the SYBR with the same results

Thanks
tc1

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The Sigma SYBR Green product that you use is for gel staining purpose. It contain DMSO which will inhibit your PCR if present too much in your PCR reaction.
You are getting inhibition effect not because SYBR Green but because the solvent of SYBR Green, the DMSO.
Reducing SYBR Green concentration from 1x to 0.1x is a correct one.

best regards
Hadrian

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Hi Hadrain
Thanks for your reply. our old SYBR green I is exactly the same product from sigma (just 4 years older!) containing DMSO and works fine. also i have tried using the new SYBR at 0.1x and while PCR product is detectable the amplification curves plateau very quickly suggesting inhibition is still occurring.

Tanya.

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[quote name='Hadrian' date='Jan 27 2006, 08:13 AM' post='38843']
[quote name='tc1' post='38561' date='Jan 25 2006, 09:59 AM']
You are getting inhibition effect not because SYBR Green but because the solvent of SYBR Green, the DMSO.
[/quote]
Hi Hadrian,

I don't think DMSO is the problem here:

DMSO formula weight is 78.13, density is 1.1g/ml, so 71ml per litre (or ul per ml if you prefer) is 1M.
Stock SYBR I is supplied in 100% DMSO at 10,000x working concentration (for gel staining anyway...) so 1x concentration is 0.1ul per ml. 0.1ul/71ul = 0.0014M => 1.4mM.

When you consider that DMSO is commonly used as a PCR enhancer at 5%(v/v) - which is 700mM - then complete inhibition of a PCR by 1.4mM seems unlikely...
http://www.promega.com/pnotes/65/6921_27/6921_27_core.pdf

all the best,

Del.

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[quote name='del' date='Feb 15 2006, 09:16 AM' post='41265']
[quote name='Hadrian' date='Jan 27 2006, 08:13 AM' post='38843']
[quote name='tc1' post='38561' date='Jan 25 2006, 09:59 AM']
You are getting inhibition effect not because SYBR Green but because the solvent of SYBR Green, the DMSO.
[/quote]
Hi Hadrian,

I don't think DMSO is the problem here:

DMSO formula weight is 78.13, density is 1.1g/ml, so 71ml per litre (or ul per ml if you prefer) is 1M.
Stock SYBR I is supplied in 100% DMSO at 10,000x working concentration (for gel staining anyway...) so 1x concentration is 0.1ul per ml. 0.1ul/71ul = 0.0014M => 1.4mM.

When you consider that DMSO is commonly used as a PCR enhancer at 5%(v/v) - which is 700mM - then complete inhibition of a PCR by 1.4mM seems unlikely...
http://www.promega.com/pnotes/65/6921_27/6921_27_core.pdf

all the best,

Del.
[/quote]

Dear Del,
Perfect calculation ! You are right.
Thanks for correcting my mistake.
But what do you think what is the reason of tc1 problem?

Best regards

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Hi,
I also though about using SYBR-Green I for QPCR instead of expensive commercial kits containing this dye. As I found in publication data, the 10 000x concentrated SYBR Green-I should be diluted 30 000 times in QPCR reaction.
Good luck,

Grzeniu

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Hi,
I think what you are seeing is SYBR Green-1 decomposition in you PCR mix which inbits taq. SYBR Green-1 is particularly sensitive to certain pH's and I guess some PCR buffers will accelerate this decomposition. To get around this I've been using EVAgreen for the past year with great success. It performs in a similar fashion to SYBR green (overlapping absorption/emmission spectra) and is very stable with v low inhibitory effects. My PCR mixes using BD titanium taq and EVAgreen now closely match commercial SYBR mixes in performance.
Cheers,
Aaron

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Hmmm... Yes, DMSO isn't the problem; SYBR itself can cause inhibition at high concentrations. I'd actually recommend using SYBR at a 1:100,000 dilution from the 10,000X stock.

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