14 replies to this topic

[Tabaluga]

Hi people,

I did a gradient PCR with several annealing temperatures and samples. The expected product length would be 200 bp. In all of my samples, regardless of temperature, I got the same single band (no smear etc.). However, this band is slightly below 200 bp, definitely !

Do you think this is the real product (but why would it be below the expected length then ?) Or how should I interpret this ?

Thanks for your help ! This issue is very important to me.....

[bob1]

Did you BLAST your primers before you sent them off to be made?  If not - there may be another binding site in the RNA or the genome somewhere that could cause this.

[hobglobin]

and how did you check it? On a standard agarose gel? and how much is slightly? A size determination on such an agarose gel is not very exact (especially with smaller bad sizes) and also other factors can influence the results (e.g. DNA amount, gel matrix heterogeneity, electric field differences)...
Perhaps you should check it twice if the same difference occurs again.

[Tabaluga]

@bob1: PrimerBLAST only gave the correct target as a result for this primer pair; besides, I took them off a publication where they have been used successfully.

@hobglobin: 2 % agarose gel. As for how much "slightly" is, I find it difficult to describe in words, I'll try to post a photo here.

[Tabaluga]

Here is the gel photo. Sorry for the bad quality, I think the bands are visible though. So the marker right "above" the PCR prduct band is 200 bp, underneath its 75 bp.



What do you think about it ?

[hobglobin]

and which one is the slightly smaller one? I could not really identify it...perhaps the far right one?

[Trof]

@Tabaluga: Did you do a negative control? Does it have the same band? (in that case - dimers)

[Tabaluga]

@Trof: I did a H2O control, nothing to see there.

@hobglobin: not sure if I understood your question correctly, but actually all of the samples have the same band, which is slightly smaller than the 200 bp marker.

[Trof]

Other bands of different PCRs are having the correct length? There is still possibility to sequence the product, to be sure what it is.

[hobglobin]

and I got it somehow wrong (thought only one of several is below 200 bp)...anyway did you ever had this then with other samples? Or is it from sequence data just expected or calculated (only)?

[Tabaluga]

@Trof: Yes, other PCR's are fine.
@hobglobin: No, didn't have this issue with other samples.


Do you think it could be the product ? Is there a possibility that the amplicon is shorter than expected for a mere technical reason, for instance ? Or would you say a certain variability is normal (but I don't think it is...)

[Trof]

No, in case other samples have expected length and there is nothing that could be changing the mobility (like different salt content, binded proteins-but those would slow down the band) it's quite unlikely your sample has the correct length.
I would sequence it.

[Tabaluga]

In the meantime I have repeated the PCR, it's still the same band size. So I guess it looks like an unspecific product then....

I'd just have two more questions:
1. The primers I took were originally designed for a real-time pcr, but I thought this shouldn't matter, or does it ?
2. Isn't it weird that the band appeared in the same strength for all annealing temperatures (50 to 62° C)? Would you not rather expect that it's stronger at certain temperatures and fainter at others (as in other gradient pcr's I did), or can it be that the temperature doesn't really matter ?

[jerryshelly1]

Very unlikely but, are you you looking at a temporally expressed gene with multiple isoforms.  With the template DNA you are using would a different isoform be expressed at this time point that is slightly shorter?  How are you generating your template DNA?  Maybe your target gene is not fully being reverse transcribed?  

1-RT-PCR primers are usually designed to have a product of 100-150Kb.  Quadruple check your gene from Pubmed with your primers by ClustalW (just to ensure there binding site).

2- Not necessarily.  Depending on the sequence of your primers, it may just have a high affinity for your gene of interest.  

*If all else fails, just redesign your primers.  I hope this helps.

[Tabaluga]

jerryshelly1, on 23 January 2013 - 10:40 AM, said:

Very unlikely but, are you you looking at a temporally expressed gene with multiple isoforms.  With the template DNA you are using would a different isoform be expressed at this time point that is slightly shorter?  How are you generating your template DNA?  Maybe your target gene is not fully being reverse transcribed?  

1-RT-PCR primers are usually designed to have a product of 100-150Kb.  Quadruple check your gene from Pubmed with your primers by ClustalW (just to ensure there binding site).

2- Not necessarily.  Depending on the sequence of your primers, it may just have a high affinity for your gene of interest.  

*If all else fails, just redesign your primers.  I hope this helps.

Hi, thanks for the reply! Actually I already ordered new primers and I'm currently trying them out. There are no known isoforms in this case. I generated the template DNA by RNA extraction from my cells and reverse transcription, how could it happen the target gene is not fully being reverse transcribed ?

As for the gradient pcr, OK that's reassuring.