recovering DNA from gel - and then secquencing it? (Sep/26/2008 )
hello,
i was wondering how to optimize DNA extraction from an agarose gel so that i can secquence a band. i use the qiagen gel extraction kit that says that it gives high yields of purified DNA apt for secquencing, neverthless, my secquences come back awful. any trick, i dunno, optimal agarose concentration, sybr vs ethidium bromide, etc?
i need to solve this problem ASAP 
thnx!
We routinely extract bands from agarose gels using the Qiagen kit and send them for sequencing, usually without problems. The only thing we do that is different from the kit is to elute in water, rather than the provided buffer (which I believe is just Tris, pH 8.0).
Are you measuring the amount of template DNA you're using? What do your sequences look like? Are you using the same primers for PCR and for sequencing? How clean is the PCR reaction from which you're isolating your band? What is the Tm of your primers? How long are they?
Personally I don't recommend dna extraction from gel for sequencing or cloning, unless you got 2 or more bands never got really good results and apparently what I see in the forums many have same problem. If you have a single band use a spin column assay for cleaning the product. If not that lucky and have several bands, then use a good low melting agarose, if possible buy band cutter (so you can work faster and get less agarose as possible). Other thing that can try is using a pair of primers that are more inside for the sequence reaction.
hi,
my secquences basically have a very high background, the lady at the sequencer here blames it on my 260/230 ratio. also, they dont resemble what im supposed to be seing at all! im sure the band im cutting is the correct one, as this worked well for the person who was in charge of this project before me (he left for a postdoc abroad and i am now in charge of this project). i have pictures of his gels clearly indicating the band and they are identical to mine.
i suspect maybe the gels here have to much bromide ethidium, i will try this weeked with sybr. i will also try designing nested primers, but this is a tricky viral sequence which will be hard to get that easily! my primers have a very low tm, maybe that could also account for the problem, but as i said before, this worked very well for the reasearcher before me (to whom i worte and doesnt have a clue of what could be going wrong and seems to be to busy to help here)
thanks a lot guys!
Another possibility - TA clone your PCR product, do colony PCR to make sure you have the right thing, then send your clone for sequencing.
yeah, thats what i thought in the first place, but i have to do over a hundred of these during this semestre and my lab dpesnt really have enough cash for all the topo kits, anitbiotics, plasmid extraction kits, etc.
advisor said just send the product to sequencing and forget all those "fancy" intermediate steps...
yeah, thats what i thought in the first place, but i have to do over a hundred of these during this semestre and my lab dpesnt really have enough cash for all the topo kits, anitbiotics, plasmid extraction kits, etc.
advisor said just send the product to sequencing and forget all those "fancy" intermediate steps...
If 260/230 is the problem. clean the reaction with EtoH precipitation. because 230 normally indicate salt contamination. i got this problem often whenever i m doing gel extraction. too much salt is NEVER good for sequencing . dissolve in H20 after that. then u good to go!
Make sure the amount of DNA is correct. Trouble can arise from either too much or too little DNA in the sequencing reaction, and is probably the single largest issue. Modern sequencers are friendlier to small amounts of DNA, but the reaction can fail dramatically with too much. You could try further purification of your DNA, but I would first make sure that you really had the amount you thought you did. You could try reprecipitating the DNA, making sure to do the 70% ethanol washes of the pellet. Salt contamination of the sample can also cause problems. Describe how you are purifying the DNA after cutting it from the gel.