I extracted plasmid DNA from an unknown bacterial specie using NEB plasmid extraction DNA kit. After runnning the DNA on 1% gel i got multiple bands of the plasmid DNA. The last band of plasmid DNA was above the 12000 band of the marker so I did restriction digestion with five enzymes. ECORI digestion yielded two cut bands one was 3.5KB in size and the other was 8.5KB in size.
I cant decipher the results of either the plasmid DNA extraction or the restriction digestion. If anyone can explain reason behind the multiple bands of the plasmid and its restriction digestion I will be extremely thankful.
I have attached the picture of the gel.
M is 1Kb marker. Lane 1 has uncut plasmid DNA. Lane 2 consists of PstI digestion, lane 3 has DpnI digestion, lane 4 consists of BamHI digestion and lane 4 and 5 have EcoRI and XbaI digestions respectively. Red arrows indicate the digested bands of EcoRI and XbaI. One is 3.5Kb and the other is 8.5Kb in size.
Plasmid extraction usually appears as multiple bands on gel. This is due to the different forms of the plasmid DNA
- Supercoiled form - This travels faster on the agarose gel due to its compact conformation
- Open circular form - This is a relaxed circular form of DNA and this runs much slower on the agarose gel
- Linear form - This is the one where both DNA strands are cut at the same place. This runs in between supercoiled and open circular form
Apart from these harsh treatment, alkaline treatment during plasmid preps denature plasmid DNA and can give rise to multiple undesired bands. Take care during alkaline lysis step and handle each and every step carefully to yield more of supercoiled plasmid form
Thank you so much Priya Soundarya Priya . This explains a lot. Although I still don't understand the digestion results. Digestion with Eco RI in the lane 5 has yielded two digested bands one 3.5 Kb in size and the other 8.5 Kb. Lets assume EcoRI has one cut site in the plasmid DNA then why two digested bands?
Mehak Ali Single restriction site will give you a band similar to the linear form of plasmid as the restriction linearizes the plasmid. There might be two restriction sites in the plasmid. and the digestion appears incomplete. I suggest you to digest 1ug of plasmid 1 unit of restriction enzyme and incubate the mixture in a waterbath/thermal cycler preheated to the incubation temperature for 3 hours. to ensure complete digestion
- Badges
- Science topic