Low yield after gel extraction - (Nov/02/2006 )
Hi I have been trying to do DNA sequencing for months but the results were either not desirable or there were no results at all! Recently, I discovered the problem might lie with my gel extraction because after PCR, i will run a gel and the band was very bright (nanodrop conc was ~ 500ng/ul). However, after using qiagen gel extraction kit, the eluent showed a very weak band in gel electrophoresis (nanodrop conc was only ~ 6 ng/ul). I use water at 60 degrees celcius for elution of the DNA and followed the procedure. What can be the problem for the low yield during gel extraction? thanks a lot for your help!
Did you make sure, that you cut out the band with as little agarose remaining on it as possible??? And was the agarose in solution or dissolved during the heating step??? What I do during the drying of the pellet is that I don't let it dry thoroughly, I wait until only a small opaque rim is still present. It increased my yield of DNA. You could give it a shot.
hi britzelbeere, thanks for the reply!
Well, i did minimise the size of agarose gel i cut out and during the heating step, i'm absolutely sure the gel was dissolved completely. I adopted the centrifuge method when using qiagen's qiaquick gel extraction kit, therefore it does not involve drying of pellet etc so i'm not too sure what you mean by that.
However, you reckon it's okay if i use the low yield i extracted and amplify that low [ ] product then send it for seq?
Thanks for your help
just one question;
Did you get the gel to QG buffer ratio correct? It is important that the chaotrophic solution is not too diluted by the gel. If the chaotropic solution is too dilute, the DNA will not bind to the silicon column. It is better to err by using too much chaotrophic solution rather then too little.
Also note that there is a limit to how much DNA that the column can bind too. Any more DNA beyond the column's binding capacity will wash through. I believe the standard binding capacity of most columns on the market is 10ug of DNA. Check your DNA is there is more then 10ug use 2 columns.
Another thing is to check the pH of your water (apart from what perneseblue said), as water gets acidic with carbondioxide from air dissolving as carbonate and thus elutes less efficiently.
When I elute DNA for sequencing (PCR purification, plasmid miniprep) I always elute in heated Tris and it doesn't affect my sequencing results, maybe give this a shot?
Two other things. I put the dna+chaotropic salt solution through the column twice -- it is easy to simply pour the flow through back into the column and spin a second time.
Then, for elution, I elute in 35 ul the first time, and then do a second elution of 25 ul into the same tube. This gives about 30-40% more DNA. You should let the elution buffer (I use Tris or TE to eliminate acidic conditions) sit on the column for a few minutes before spinning.
As described in the kit, if your DNA is <500bp or more than >4kb, should include equal vol isopropanol.
phage434's suggestion works too (running the QG sol with DNA twice through the column), but as perneseblue pointed out, the column has a max. binding capacity. But if you're working on DNA from (ONE) well, that's probably ok.
For better yield or elution, could follow vairus's suggestion, i.e. use preheated TE for elution. Normally, I'd add H2O or TE first, then heat at 50C for 5 to 10 min. Then spin to elute.
Are you saying that you add the water to the column first and then heat up the entire column to 50C? I've always just heated the water before adding it to the column...have I been doing it wrong or is your way a modification that works better?
Both ways work fine for me. But I prefer the latter, i.e. add, then heat. There's no right or wrong... just to "accelerate" the dissolution of DNA in the column into the solution by mild heat/temperature.
In how much volume are you eluting your DNA? I like to use the Zymogen colums that are made to elute your DNA in as little as 6uL. I used to have that problem when using the QuiAgen Kit since their colums were big and you have to elute in no less than 700uL I belive.
Also, you can try to do the Freeze and Squeeze method to elute your DNA directly from the gel into a tube. Its a little more durty but it works.