I did ask for the most efficient (higher recovery, good quality) but not fastest.
Recovering DNA from a gel is not going to be efficient, as you'll loose a lot during the process, especially if you product is 105bp or less. However, it is possible to minimize the loss. Firstly, run the gel at a low voltage, I think you can go as low as 40V, but 50V has worked OK for me. Secondly, when cutting out the band work quickly an minimize exposure to UV, the more UV the less DNA! Thirdly, During the melting procedure keep the heat block at as low temperature as possible, 50C is ok, I think most kits recommend 50-65C.
Another tip is when you know you have to run a gel cut out simply add a few more cycles to the PCR giving you more product.
In relation to kits, all seem to give about the same result. We use Wizard SV
If you can avoid a gel cut and can simply run a PCR cleanup I'd recommend chargeswitch, as bead recovery seems to be a lot better the spin column based kits.
Hope this helps and regards
I have had pretty good success with Wizard® SV Gel and PCR Clean-Up System by Promega. Always good quality, the yield is dependent on what kind of DNA you are trying to purify (I.E single plasmid band etc)
http://au.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/
Recovering DNA from a gel is not going to be efficient, as you'll loose a lot during the process, especially if you product is 105bp or less. However, it is possible to minimize the loss. Firstly, run the gel at a low voltage, I think you can go as low as 40V, but 50V has worked OK for me. Secondly, when cutting out the band work quickly an minimize exposure to UV, the more UV the less DNA! Thirdly, During the melting procedure keep the heat block at as low temperature as possible, 50C is ok, I think most kits recommend 50-65C.
Another tip is when you know you have to run a gel cut out simply add a few more cycles to the PCR giving you more product.
In relation to kits, all seem to give about the same result. We use Wizard SV
If you can avoid a gel cut and can simply run a PCR cleanup I'd recommend chargeswitch, as bead recovery seems to be a lot better the spin column based kits.
Hope this helps and regards
Hi
two more tips to avoid UV exposure...
1) if the DNA product is in good amount some other dyes cane be used to visualize the DNA without UV exposure
2) the product can be loaded in two lanes side by side....in one lane load less amount and use it for staining with EtBR and to mark the region of fragment during UV exposure...the adjacent lane can be loaded with more amount and used for isolation without the UV and by aligning it with the adjacent lane....
bye
Dear Christopher, we share experiences on running the sequencing gels, although I did for RNAse protection assays and not for sequencing, and the gels were the large 90 cm EMBL with a glass heating plate where you pumped H2O at 50ºC into. I also used 35S and 32P, even some 33P..... but I am having serious problems with the kits to purify DNA (actually fragments from plasmids). At the time I got a >90% recovery with a device from Schelicher & Schüel that was called BioTrap or EluTrap (depending on the side of the Ocean you where in). When I state 90% I mean that I have put 10000 counts of labelled DNa (or RNA ) and recovered 9000. My question is how do you do this today?
I use to purify DNA from the kit available in the market. Like, QIAgen, Invitrogen, Life Technologies etc. But I find QIAgen, the most suitable one.
Try to cut as less agarose as possible.
Take the excess of the disolving buffer.
Desar Jose,
I did nearly all procedures for eluting DNA from agarose or acrylamide gels myself during my laboratory life, including the "biotrap" approach that you mention.
Today, we almost always make use of binding DNA to small glass particles. I recommend just to by commercial "glas milk" (It is absolutely no fun to make glass particles yourself!). The rest is very easy and uses normal solutions that must not necessarily be bought in expensive kits.
I recommend the following procedure (yields are normally around 80 %).
* cut the band from the agarose gel (the normal one!)
* add 3-5 volumes 6 M sodium iodide solution *
* melt for 5 min at 55 °C
* add 5 µl commercial glass milk (this is sufficient for 5 µg DNA– you never need more glassmilk!); wait for 5 min at room temperature; shake from time to time
* centrifuge for 5 sec
* wash 3 x with 0.5 ml 20 mM Tris-Cl, pH=7.4, 100 mM NaCl, 1 mM EDTA, 50 % ethanol
* resuspend in 10 µl 10 mM Tris-Cl, pH=8.0, 1 mM EDTA
* elute for 3 min at 45 °C
* centrifuge and use the supernatant for your cloning work or whatever
Sodium Iodide solution is made that way:
* Dissolve 0.75 g Na2SO3 (sodium sulfite) in 40 ml water.
* Add 45 g Na-iodide and dissolve.
* Filter the solution and store in the dark.
This works nicely and can be done in parallel wth as many fragments as Eppendorf tubes fit in your centrifuge.
Best wishes - Johannes.
Of course, it is depend on your plant. Also your method, either manually or kits?
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