Circular DNA makes a smear when running in agarose gel, but the digested linear form doesn't. I am not talking about covalently or supercoiled bonded.
I'm not sure how in your question you have ruled out supercoiling. Intact circular plasmid DNA always runs to some degree as a smear; this is supercoiling. To confirm, one usually cuts a bit at a single site and the smear collapses to a single sharp band usually higher than the smear.
I think that the isolation did not work so well, and it is ruined or it Starts to be degraded. You also gas to pay attention on How big are the fragments of the smear. If they are concentrated at less than 3-4kbp....I would suggest to start with another isolation
Could be different supercoiled forms; really large circles get "hooked".
1. During isolation, u please maintain the incubation time when u treat with SDS.
Thats the crucial part of it.
2. U have to give more time for RNAse digestion.
3. Before running u do a purity check with NanoDrop.
i think that should avoid your problem.
I use RNAse every isolation, furthermore when i digested same plasmid with a RE, the smear's not seen in gel.
Dear Mr. Nami...
If u could post a gel image I can help with that. this is a peculiar problem i have come across with.. If u could post the gel image.. it would help find an answer.
Using DNA columns may resolve your problem...
Once I had this problem because of change in the pH of the suspension buffer i used for suspending the DNA. I think U better check with the chemicals and buffers used for isolation.
Of which size are your plasmids/bacs? My experience with BACs is, that they are really big (200-300kb) and are easily sheared by incautious handing. You will then get a smear on the gel.
It could be too concentrated, the there is always a huge smear.
I dont think it is RNA, RNA smera are usully at like 250bp and easily detectable.
It is also possible that you have ethanol contamination from your washes in the last steps. If this is the case you might have trouble loading your DNA into the wells, the liquid is not very dense and tries to come back out.
The best way to fix this, is to run it throught a Sepadex column, which is also good for desalting and any other downstream applications as it really purifies the DNA. I use it fopr all of my isolated cDNA/Plasmid DNA/Ligation. Protocol below (but I still think it might just be too concentrated, because of yourde the concentration is lower after restriction)
Prepare Stock solution
1x TE buffer (10mM Tris-HCl, pH 7.5, 1 mM EDTA)
• Suspend 33.33g of Sephadex G-25 in 500ml 1x TE buffer
• Equibrillate o/n @ 4degrees
• Let Sephadex settle down and take off supernatant (you will lose some, but don’t worry)
• Resuspend in 500 ml 0.2 M NaOH and leave for 20min @ RT
• Take off supernatant and wash 5x in 500 ml MQ-Water (you will lose some, don’t worry)
• Resuspend in 250ml 1x TE buffer
• Autoclave
Desalt
• Suspend prepared stock solution and transfer 200 ul into PCR tube (keep sterile
• Punch a very little hole into the bottom of the tube with a #25 syringe
• Spin with open lid 1 min @ 2,000 x g
• If filtrate is clear, add your sample (up to ~30 ul) onto the sepadex and spin 1min @ 2,000 x g with open tube lid
I'm not sure how in your question you have ruled out supercoiling. Intact circular plasmid DNA always runs to some degree as a smear; this is supercoiling. To confirm, one usually cuts a bit at a single site and the smear collapses to a single sharp band usually higher than the smear.
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