problems with qiagen gel extraction kit - (Feb/21/2010 )
I am using qiagen gel extraction kit for purification of my digested vector and insert from 1% agarose gel, I try exact protocol of kit for 4 times, my fragments in gel very sharp and I cut exactly dna containing part of gel and use 400mg from gel for each column. but after elution in nuclease free DW or kit elution buffer I get only 10-20 microgram/microliter of purified dna, I dont know what is wrong. in addition the kit is a new one with 8-9 months to its expiration date.
any help is appreciated
It is tough to judge if you're having recovery difficulties without knowing how much DNA you started with. Is 10-20 ug/ul 40% recovery? 80%?
How much water are you eluting in? If you're using 50 ul, is 500 - 1000 ug of DNA not enough for you to proceed? What are you intending to use the recovered fragments for?
Is it necessary to use a 1% gel?
Can you push multiple aliquots of dissolved gel slices through a single column to maximize the concentration of the eluted sample?
How old is the PE-Buffer? If it was prepared a little while ago, probably the EtOH evaporated. Maybe your DNA get flushed away during the wash step.
But for what do you need the DNA? For ligation? Doesn't it work? If all your downstream-application work I wouldn't bother to much.
You can also try to contact the Qiagen Support, they are usually very helpful.
I've frequently used the gel extraction kit. It gives really poor yields, usually less than 50% of what was loaded. If you're using the Qiaquick kit, you can elute in as little as 30 ul. If you use the Minelute kit, however, you can drop the elution volume down to 10 ul. The drawback of the Minelute kit is that you shouldn't try to purify a piece greater than 4 kb. Anything smaller than that works pretty well.
When I do gel purifications, I load as much into one well that I can, then cut out two adjacent bands in one plug and purify them together.
And are you sure you're getting 10-20 ug/ul or is it 10-20 ng/ul? My purifications are usually in the ng/ul level. If you're using the vector for ligations, you usually only need about 100 ng of vector in the reaction for a successful ligation, so even if you are getting ng/ul levels, 5-10 ul of that is good enough for a ligation reaction.
fishdoc on Feb 21 2010, 10:54 AM said:
When I do gel purifications, I load as much into one well that I can, then cut out two adjacent bands in one plug and purify them together.
And are you sure you're getting 10-20 ug/ul or is it 10-20 ng/ul? My purifications are usually in the ng/ul level. If you're using the vector for ligations, you usually only need about 100 ng of vector in the reaction for a successful ligation, so even if you are getting ng/ul levels, 5-10 ul of that is good enough for a ligation reaction.
Hi
Thank you very much for replies, I start with 10-15 microgram digested plasmid DNA, I plan to digest out a fragment from T vector and clone it into another vector, my final yeild of vector after gel purification is 10ng/ul and for insert I get 15-20 ng/ul. I do try ligation with this fragments and I have cheched my ligase with a control, it worked but in my own ligation reaction I got nothing. for having 100 ng vector I should add at least 10 ul vector and in a 1-3 ratio the insert is 15 ul adding enzyme and buffer my final ligation reaction is about 30 ul that is too diluted, I think ligation reactions are better to be in more smaller reactions like 10ul, so if I can increase my recovery I might be successfull in ligation.
sara.r on Feb 22 2010, 01:02 AM said:
Thank you very much for replies, I start with 10-15 microgram digested plasmid DNA, I plan to digest out a fragment from T vector and clone it into another vector, my final yeild of vector after gel purification is 10ng/ul and for insert I get 15-20 ng/ul. I do try ligation with this fragments and I have cheched my ligase with a control, it worked but in my own ligation reaction I got nothing. for having 100 ng vector I should add at least 10 ul vector and in a 1-3 ratio the insert is 15 ul adding enzyme and buffer my final ligation reaction is about 30 ul that is too diluted, I think ligation reactions are better to be in more smaller reactions like 10ul, so if I can increase my recovery I might be successfull in ligation.
I usually do ligation reactions in 20-30 ul volumes. I've done 10 as well, but haven't seen much difference using the larger volume. I think DNA concentration is more important. I use NEB T4 ligase, and their FAQ recommends having something like 1 to 10 ug/ml (ng/ul) of DNA for the reaction. If available, check an FAQ for your ligase to see if the optimal concentration is given.
"To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation."
http://www.neb.com/nebecomm/products/faqproductm0202.asp#346
Different enzymes may vary, though.
Do your ligations with 10 ng of vector and about 10 ng of insert (1-3 x the molar amount) in 10 ul. Higher concentrations favor multimers, which do not transform. You want circular constructs, favored by low concentrations. But the yield of your gel extraction is so very low, I'd be concerned that there is something very wrong. Are you spinning the columns dry after emptying the buffer PE flow through? What is your elution volume? You might try heating your elution buffer (but I doubt that is the problem).
I just want to check that you really meant 10 ng/ul in your earlier posting, not (as you said) 10 ug/ul. The latter would indicate a real problem since the column capacity is only 10 ug or so, and would probably indicate a bad contamination problem, rather than DNA.
I would recommend checking the DNA quality and amounts by running a gel with some known concentration bands and comparing band intensities. With some 2x serial dilutions of your DNA, you can accurately pin down amounts with much better assurance you really have DNA than with a spec.
I know it might sound odd, but I use only one colums even of I have 1-2 gms of agarose gel. I have comapred recovery rate using 1 column and also doing it in 3-4 columns. A single column from Qiagen kit works even for larger gel pieces with large amounts of DNA.
Can you estimate how much DNA is in the gel when you run it? If you start with a low amount you will lose proportionately and might end up with nothing.
If you get 10-20ug/ul, then its a lot of DNA. Its probably ng/ul. EVen then it is sufficent for ligation reactions.