PCR band slightly BELOW expected length ?! - (Jan/14/2013 )
@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...)
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.
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 ?
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.
jerryshelly1 on Wed Jan 23 18:40:31 2013 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.