12 replies to this topic

[Maddie]

Hi All,

I am writing this post on behalf of a colleague who has problems with her SYBR Green real time assay. I'm not familiar with SYBR Green so please don’t shoot the messenger.  
She is using ITS primers to amplify all kinds of fungi, her amplicon is ~600bp. I know it's quite large for such an assay but she is using a published protocol. Problem is, her amplification efficiency is never above 63% and her melting curves are all over the place, even with her standards that are pure A. niger. I include a picture that should speak for itself (these are only standards).
Does anyone knows what's happening here? Why are there so many peaks in the melting curves? What can she do to improve her efficiency (does it really matter)?
Thank you.
Maddie

Attached Thumbnails

• 

[Maddie]

I should also mention that these standards are made with the PCR product she got when targeting 600bp of A. niger, so it should be good quality DNA and rather homogenous.

[Maddie]

Nobody can help?  

[Adrian K]

Are you sure she is doing the right thing?
I did ITS before... but we are not using real time PCR to do it. Just normal PCR and sequencing.

[Maddie]

adrian kohsf, on 24 February 2011 - 11:03 AM, said:

Are you sure she is doing the right thing?
I did ITS before... but we are not using real time PCR to do it. Just normal PCR and sequencing.

Well, I've never checked closely what she does, no   . I tend to trust her when she says she is using the published protocol.   I mean, in this assay there is not much room for improvisation I think. The master mix is ready to go so basically only the primer concentration varies, right? She ran the qPCR product on an agarose gel (from the exact same samples I'm showing above) and got  a single clean band for each standard. I am baffled  

I need to check the ITS sequence to see if there are "domains" in there, like GC rich fragments surrounded by regular sequences. Could that explain?
And what can she do to increase her PCR efficiency? The author claims he as 90%, but she never got more than 63.

[Adrian K]

From what I understand regarding ITS region, it is a repetitive, inter-transcribed spacer region. I'm sorry I had never hands on in real-time before can can't help much.
I don't remember that it is GC rich either...

[Maddie]

We're in the same boat then  

[Adrian K]

Maddie, on 25 February 2011 - 08:17 AM, said:

We're in the same boat then  

Same boat? Let's sink together then...  

Coincidently, vtoday I asked a manager from my supplier's company regarding a job of technical specialist application: he told me at least I should have experienced in using Real-time PCR or else I should have no chance in his company... sigh...

Now, where should I get money to fund myself now... boss got no more money to fund me....

[Maddie]

adrian kohsf, on 25 February 2011 - 08:57 AM, said:

Maddie, on 25 February 2011 - 08:17 AM, said:

We're in the same boat then  

Same boat? Let's sink together then...  

Quote

Coincidently, vtoday I asked a manager from my supplier's company regarding a job of technical specialist application: he told me at least I should have experienced in using Real-time PCR or else I should have no chance in his company... sigh...

Now, where should I get money to fund myself now... boss got no more money to fund me....

Hmm you could actually start with SYBRgreen, the kit is relatively cheap compared to Taqman. Do you have an instrument?

[Adrian K]

LOLx Maddie, but I got no money left... haha

[Maddie]

adrian kohsf, on 25 February 2011 - 12:37 PM, said:

LOLx Maddie, but I got no money left... haha

Hmmm that's a slight complication  
I could give you a recipe to make a home-made thermocycler with pans and a stove but real-time instrument is beyond my abilities  

[Maddie]

BTW, I'm onto something. Looks like the taq from the ABI SYBR green kit isn't a hotstart, sooo maybe all these crazy peaks I see in the dissociation curves are unspecific products?? AB calls the enzymne Fast Taq and includes an activation step but since it is only 2 seconds... ..I'd say this is a regular enzyme.
Will try the Sigma kit that does use a hotstart Taq.

[Adrian K]

Maddie, on 28 February 2011 - 07:28 AM, said:

adrian kohsf, on 25 February 2011 - 12:37 PM, said:

LOLx Maddie, but I got no money left... haha

Hmmm that's a slight complication  
I could give you a recipe to make a home-made thermocycler with pans and a stove but real-time instrument is beyond my abilities  

I like to have that recipe.
Hmn... what about 3 water bath with different temperature on?  

Quote

BTW, I'm onto something. Looks like the taq from the ABI SYBR green kit isn't a hotstart, sooo maybe all these crazy peaks I see in the dissociation curves are unspecific products?? AB calls the enzymne Fast Taq and includes an activation step but since it is only 2 seconds...  ..I'd say this is a regular enzyme.
Will try the Sigma kit that does use a hotstart Taq.

I don't think the hotstart will have such a great effect on it, but I have to say I really not sure... perhaps she can write to the author to ask about that and maybe let us know what happen..