I'm working on a 35bps regulatory part from the iGEM Registry of Standard Biological Parts. My band size should be exactly 35bps when run against a 50bps ladder. But it shows to be ~100bp. The PCR cycles (25) and extension time are optimized for this part. But on a 2% agarose gel I don't get my desired band size. Any idea whats gojng wrong?
I agree with the comment that PAGE provides better resolution
I guess the iGEM standard part was sent to you in a plasmid, that reduces the chance of getting non target amplification. it is still possible that there is some non-target amplification.
usually primers are designed to amplify more than the target region, which is why I urge you to check the primer design by matching the primers to your DNA source; personally, if i were to amplify a promoter region, i would make the target size manageable(say around 100bp?), also your target might contain a DNA sequence motif that would have forced the primer to be designed to give more than 35 bp of DNA as a PCR product.
just a thought, by the general PCR design rules primers need to be a minimum of 18 nt, that would mean sum of forward(18 nt) and reverse primers(18 nt) alone would give you 36 nt. you might not be able to distinguish between PCR product and primer dimers even by electrophoresis in PAGE.
Please don't mind me asking, I am just curious.
Why would someone take this approach of cloning a 35 bp DNA sequence?
- the sequence is already known since it is a iGEM part. so, i guess you don't intend to sequence it.
- according to the iGEM standard, your regulatory region will have flanking restriction sites, you could clone your ORF directly into the plasmid in which you received your regulatory region.
- these days one can easily avail PCR primers of more than 50bp. primers of these lengths would allow us to directly clone the ORF under the regulatory region.
- or longer primers can be used to perform SOE-PCR.
You must use polyacrylamide gel electrophoresis, which suitable for your DNA sample size 35 bps ( suitable for small sizes) because it is pores very small and hinder the sample , while the agarose gel is perfor for DNA sizes 100bps and more
what size are your primers to get a 35bp amplimer please. Very short primers often produce unexpected bands and an additive may be needed to make them more specific
I agree with the comment that PAGE provides better resolution
I guess the iGEM standard part was sent to you in a plasmid, that reduces the chance of getting non target amplification. it is still possible that there is some non-target amplification.
usually primers are designed to amplify more than the target region, which is why I urge you to check the primer design by matching the primers to your DNA source; personally, if i were to amplify a promoter region, i would make the target size manageable(say around 100bp?), also your target might contain a DNA sequence motif that would have forced the primer to be designed to give more than 35 bp of DNA as a PCR product.
just a thought, by the general PCR design rules primers need to be a minimum of 18 nt, that would mean sum of forward(18 nt) and reverse primers(18 nt) alone would give you 36 nt. you might not be able to distinguish between PCR product and primer dimers even by electrophoresis in PAGE.
Please don't mind me asking, I am just curious.
Why would someone take this approach of cloning a 35 bp DNA sequence?
- the sequence is already known since it is a iGEM part. so, i guess you don't intend to sequence it.
- according to the iGEM standard, your regulatory region will have flanking restriction sites, you could clone your ORF directly into the plasmid in which you received your regulatory region.
- these days one can easily avail PCR primers of more than 50bp. primers of these lengths would allow us to directly clone the ORF under the regulatory region.
- or longer primers can be used to perform SOE-PCR.
1. What is the purpose for your PCR? If you only for detection of target gene, You should use the long primer to achieve longer PCR product.
2. As a said above, designing long primer and increasing annealing temperature for PCR.
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