PCR bands on gel - (Dec/14/2015 )

Hello all,

I am trying to amplify a 1650bp fragment.

However when I PCR amplify this fragment I get a piece on gel that is right below the 1500bp marker.

Is this normal?

(usually my bands are pretty much spot on, at the correct place).

Several possibilities:

1. Correct size, but band seems too small because of the gel (gel ran inhomogeneous, e.g. outer lanes where the size ladder is often placed, did not run at same speed but slower)

2. elongation time of PCR is too short

3. Other allele/genotype or mutation with different size (depending on template this can be possible or not)

4. Unspecific amplification

5. Incorrect size estimation (if e.g. band is just to blurry or broad)

...

hobglobin on Mon Dec 14 20:32:13 2015 said:

Several possibilities:

1. Correct size, but band seems too small because of the gel (gel ran inhomogeneous, e.g. outer lanes where the size ladder is often placed, did not run at same speed but slower)

2. elongation time of PCR is too short

3. Other allele/genotype or mutation with different size (depending on template this can be possible or not)

4. Unspecific amplification

5. Incorrect size estimation (if e.g. band is just to blurry or broad)

...

I attached a picture.

The bands should be +- 1650-1700bps and you can see they are below 1500!

It seems like the PCR worked, but I can not figure out how come they are so low.

Elongation of PCR: should not be a problem, I have 2 minutes.

Other allele genotype: no problem, its from a plasmid, just 1 gene

Unspecific amplification, seems weird, I use a primer that has 18bps to use (+RE at the end)

incorrect size estimation? I doubt it, it looks clear to me.

Two Sanger sequenincg reactions would immediately tell you. Use each of the two PCR primers individually to sequence from either end. Do you have other evidence that your template is what you think it is?

If it's a plasmid some secondary structure might influence running speed...

I wouldn't be concerned about the size discrepancy assuming your primers and template are correct. Overloading, secondary structures, methylation (?), etc can lead to migration anomalies. Electrophoresis (both DNA and protein) is not always a robust measurement of size.

I routinely (i.e. hundreds of times now) amplify a 2kb insert, but on a gel it always runs ~1500bp. Sequencing has shown every single time that it's the perfect 2kb insert. 

Sequence it from both ends and you'll have your answer.