I've conducted a quality check of my pBR322 using agarose gel electrophoresis. I got my supercoiled form plasmid band at around 2500-3000 bp and open circular form band at around 6000 bp. Why didn't I get approximately 4000 bp instead (since pBR322 has 4361 bp)?
Hi there,
If you don't get any band corresponding to the actual size of the vector it means there is no linear form of the vector (or at least no detectable amount) in your plasmid prep.
Agar (Etymology; originally from Malay or bahasa Melayu as agar-agar) is a polysaccharide from red algae such as Gelidium elegans and Gracilaria vermiculophylla. Agarose has some amount of pyruvate and sulphate residues in the component sugars (D-galactose and 3,6-anhydro-L-galactopyranose). Then, effect of pyruvate and sulphate residues may sometimes appear.
Polyacrylamide gel (PAG) may have inversely positive charges together with strong hydrphobic nature in the gel.
Therefore, the electrophoresis of DNA and RNA by agarose gel turns out to be difficult unexpectedly.
Then, please use our HPLC-Soap-SEC developed for hydrophobic proteins to check the precise molecular weight (please see file "SEC column 300A silica").
The absence of a ~4000 bo band is not surprising since it represents a linearised form of the circular pBR322 plasmid which should not be present in intact DNA This means that your plasmid DNA prep is very good occuring in supercoiled and open-circle (relaxed, some single strand breaks) configurations as mentioned before. A linearised form can be obtained by digestion of plasmid DNA with a restriction enzyme having a single recognition site in DNA. On a gel, both 2500-3000 bp and 6000 bp bands should disappear after the digestion. Instead, a band of ~4000 bp should appear. Such an experiment is elegant and suitable to students' practicum.
On a Agarose gel, both 2500-3000 bp and 6000 bp bands should be disappeared after the enzymatic digestion, that is why, a band which contains about ~4000 bp plasmid DNA should be appeared. Therefore, the absence of a ~4000 bp plasmid DNA band is not surprising at all.
As Ales Kovarik explained you may have two or three bands of different molecular weigh when the plasmid in not digested, but when you do performed a digestion with one restriction enzyme you should obtain a single band of approx. 4000 bp. Another tip is you can by a supercoiled molecular weight marker special for plasmids.
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