PCR product purification - (Apr/01/2004 )
In my point of view this doesn't look like a problem with the kits you are using, but something to do with the gel conditions. Please, double and triple check that your buffers and gel conditions are at least sufficient to obtain a clean sample. GEL ARE TRICKY!!!!! A very good alternative is to ask a collegue to give you some of his/her sample (positive control, or you should have some allready) and check out how this is shown in the gel, and what is the purification yield, if any!
Try this if it seems logical to you, and tell me the aftermath
if you have your band at the expected size without other nonspecific bands, try EtOH precipitation if you are kits-phobia !!
hello gentle man,
i used to use promega whizard pDNA purification kit.
i hav used it to purify 256bp DNA product.. so no soubt to follow it.
u have to give more incubation time with the purification resin and hav to getly mix it for more than 5 min.
all the best
Qiagen's spin column formats sometime have this problem that bother you so much and you just don't know what's going wrong. Try Epoch Biolabs' GenCatch PCR clean-up kit (epochbiolabs.com), it's a better kit than Qiagen's in both consistency and price in our lab.
usally we purify directly from excised gel fragment using spin columns. this is followed by EtOH precipitation & re-dissolution.
that has always worked wonderfully without giving any problems. ever tried that?
I use Jetsorb from Genomed. It works every time with small and bigger fragments.
for PCR product purification used for cloning, phenol/chloroform's classical method is ok. I think kits are unnecessary.
I use the Qiaquick PCR purification kit all the time without having any troubles. Maybe your PCR did not work. Otherwise you can use a PCR puirification kit from Roche.
First look on agarose gel (2%) that you have PCR product of interest.
If you have low concentration you can pool some samples together (same kind of PCR product of course )
I normally use the PCR purification kit (Jet quick) from genomed Incorporation. I thinkl that one is working just fine.
I have purified PCR product that is 150bp.
I hope that you soon will purify youre sample succesfully.
Don't forget that many of the kits elute the purified PCR product in water or buffer. This generally dilutes your product, especially for gel bands! 100 ng of DNA will give a nice, bright band if the initial well in the gel wasn't very wide. Bind that 100ng to a membrane and elute into 50 ul of buffer, and you've got a concentration of only 2 ng/ul! Load 5 ul on a gel to check for product/quantify, and you probably won't see a thing. Eluting in less than 50 ul increasaes your concentration, but you lose some product in the process.
So, if you don't see product after cleaning, and think you've lost your product, my suggestion would be to ethanol precipitate, preferably with a co-precipitant such as PelletPaint (Novagen). Then you can see if you have a product, cause if you don't you also won't have a pellet, and you can get your concentration back up to where you can see it on a gel or quantify it by spec.
Just my 2-cents worth.