6 replies to this topic

[inza]

I have extracted plasmid DNA using standard method which use alkaline lysis and i got a lot of precipitate after ethanol precipitation. After solute with TE buffer , I checked the plasmid DNA with 1 % Agarose gel and unfortunately there was no band can be seen. I found out from the picture taken that there somewhat lots of smear in the well of the gel. Is it possible that the plasmid DNA is not separated? Or, if there any other possible reason occurs?

[Ihab Ismail]

I have encountered similar situation. The buffer might be old. I suggest making a fresh one.  

[Eddie]

May be your DNA is too big and closed the gel pore?
Say me more detailly concern your done work.

[danielxu]

I think you may have your plasmid DNA extraction imcomplete since you've got lots of ppt in the ethanol precipitation step. Aliquiot more to other eppendrof for lysis is better.

[inza]

Thanks for few suggestions received. I have renewed the TE buffer and extraction buffer. But still the similar thing occured. I will try other plasmid miniprep method. Just unable to figure out the smear in the wells. Maybe the plasmid extraction is not completed. I found out though that after boiling the extracted plasmid and ETOH precipitation once more have reduced the smear in well and maybe i will try the the boiling method for a change.

[Ihab Ismail]

Hello again. I have been trying the regular plasmid miniprep for my binary vector (maniatis manual book). What I meant by the buffer is the running buffer (TBE,  TAE, etc).  
Good Luck

[inza]

Thanks again for all the suggestion that have been given. Finally I got the bands and thank God that the only problem is that im using old TBE buffer and I renew it. The expected band can be seen clearly. Thanks .