Plasmid DNA extraction - Extracted plasmid cannot be seen in agarose gel (Jul/29/2002 )
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?
I have encountered similar situation. The buffer might be old. I suggest making a fresh one.
May be your DNA is too big and closed the gel pore?
Say me more detailly concern your done work.
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.
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.
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).
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 .
Hi! I´m looking for a simple, fast and cheap protocol for plasmid DNA extraction.
At this moment I'm using only alkaline lysis but I would like to get rid of the contaminant proteins and RNA. I've already obtained nice bands, but not all the time. So, I would like to improve a little the protocol.
Usually when you have a "large" pellet in your precipitation, it could be a sign that your DNA is contaminated with chromosomal DNA and protein, i.e. that your plasmid prep was not clean. You should make sure you have selected correctly for your plasmid and try your prep again. Many people use the qiagen spin preps because they are fast and relatively cheap. Check your 260/280 ratio and see how clean the dna you are purifying really is, it will probably explain the "smear" you are encountering.
Thank you tap14!!!
You´re right, I'm going to check that out!!!
If I keep having any trouble I'll let you know!!!