Hi,

fluorescence in wells of agarose gel could indicate impurities. please check the concentration and purity ratio of fosmid DNA.  

try digesting the fosmid with restriction enzyme to confirm it hasn't moved due to high molecular weight

you could use wide bore pipette tips to reduce shearing.

regards.

Thank you Vamsi Bharadwaj SV for answering 

Actually I did not shear the fosmid in order to check the integrity before sequencing.

So for the fluorescence in wells you do not think that it represents a large DNA ?  

What is the percentage of agarose you're using for your gel? I used to work with very long fragments (30kb), and performed 0.5% agarose gel.

The smears are more concerning in fact, indicating DNA degradation, with multiple possible source of contamination (TBE, problem with fosmid purification...)

Hi again,

There are high range ladders which could be used to measure DNA sizes of about 49 KBp, so it is highly unlikely that the fluorescence in the wells of gel is your fosmid, if you have run the gel long enough at appropriate voltage (2.5-3V/cm for 1.5h for example)

regards.

Dear Thibault Caradec  

Thank you for your interest. Actually I am using 0.8% agarose gel. In case of using 0.5%, what should I change in the other parameters ( voltage and time) ? 

Dear Vamsi Bharadwaj SV

I will take your remark into consideration, Thanks again :)