Dear Fadillah,

It could be caused by DNA degradation. Bacteria contain endogenous nucleases that can rapidly degrade DNA and produce faint blurred digests. Failure to chill and wash the bacteria promptly when harvesting the broth culture is a common cause of degradation, and the plugs must be prepared smoothly and rapidly to assure that endogenous nucleases are promptly inactivated by the proteases and the EDTA. An other thing that you can do is to add thiourea in the running buffer.

An other cause could be the incomplete lysis and/or incomplete protein digestion.  Incomplete digestion can occur if any of the reagents used to prepare the DNA (proteinase K, EDTA, detergent) are not completely removed, resulting in loss or inhibition of the restriction enzyme; the extensive washes in water and TE are essential to removing all traces of these agents from the plugs. In addition, some types of agarose are reported to contain inhibitors for certain restriction enzymes. If the cell lysis is believed to be adequate, but the restriction digestion is incomplete, the plugs can be washed thoroughly with TE and redigested with fresh restriction enzyme.

Hope this help,

Simon

Hi.

I agree with Simon Lévesque. If you splitted your plugs before the run you can try again. Sometimes, for me the 2nd run was better than the 1st one. I can not explain this phenomena as the plug was just devided in 2 parts.

Thanks Simon and Toni for the suggestions. Will try them out soon.

Apart from the stated lanes, which other lanes do you suggest need repeating for a better process during analysis?

Hi Fadillah, if if it not too much work, I suggest to you to repeat all the gel in order to compare with your new protocol.

Good luck,

Simon

Thank you Simon. 

IS there a possibility that you had less cells in those plugs? Also, are they different than rest of the strains? Do they have thicker capsule? then they might need different treatment during lysis step.

Hi Bhaswati, the adjusted absorbance of the culture was around 0.9 at 610 nm. All of the samples are S. aureus; possibly of different strains. I am not sure about their capsule thickness but all were incubated overnight in EC lysis buffer. For the next run, i am trying to increase the absorbance to 1 nm and increase the plug washing. 

i think there is some problem with the uniformity of gel staining. Either the gel was reused or you need to restain with Eth Br. Reload on a new gel