Huge DNA loss after purification - (Mar/19/2006 )
Hi all, I linearized a vector (~8kb) purified from Miniprep (qiagen), did a digest, ran it on gel (to chk for complete digestion) and gel purified it (qiagen kit). I used ~300ng/microlit for digestion, the gel bands looked ok, but somehow after the purification step i get only 12ng/microlit!!
So I thought it was because the vector was too big since qiagen recommends that their kit be used for up to 4kb.
Then i used a different kit from Zymo (DNA clean and concentrator) which is suitable for DNA up to 23kb. this time i didn't run it on gel since i know that there is complete digestion. Still only 12ng/microlit...
Ok so to if size is really affecting the yield... i double digest the vector to release a ~3kb insert. gel purified... and at the end of it, only 5ng/microlit!!!! Thats barely anything. Seems to be nothing to do with size!
anybody knows whats wrong? I added ethBr to my gel before running it... i use to do post-staining in my previous lab.. i wonder if it has anything to do with that? 
It's happens more, I can tell you.
You can try to dissolve the agarose in the buffer at 37C, this also works and the purification yield often gets higher. But I know your problem and I just accepted it and often load more on gel for purification because I lose some.
Other people? 
me too, add more before purifation, you will get more...haha..
good luck.
So I thought it was because the vector was too big since qiagen recommends that their kit be used for up to 4kb.
Then i used a different kit from Zymo (DNA clean and concentrator) which is suitable for DNA up to 23kb. this time i didn't run it on gel since i know that there is complete digestion. Still only 12ng/microlit...
Ok so to if size is really affecting the yield... i double digest the vector to release a ~3kb insert. gel purified... and at the end of it, only 5ng/microlit!!!! Thats barely anything. Seems to be nothing to do with size!
anybody knows whats wrong? I added ethBr to my gel before running it... i use to do post-staining in my previous lab.. i wonder if it has anything to do with that?
I don't think the ethBr has something to do with your problems ( but I do think that post-staining is much less of a mess
)- maybe 300ng is too much for the column? What did you use to elute you DNA- destilled water? Try with warm TE buffer instead if it doesn't affect you following steps. That helps sometimes.good luck!
)- maybe 300ng is too much for the column? What did you use to elute you DNA- destilled water? Try with warm TE buffer instead if it doesn't affect you following steps. That helps sometimes.good luck!
So I repeated everything (using qiagen) but this time half of the samples were not exposed to UV at all when i was cutting the gel out for purification... haha.. it made a 2ng/ul difference!
I use 2 columns for 300ng... and yeah I elute with milliQ water. I will be using the purified fragment for ligation. Will TE buffer affect it? My supervisor use to advise me against it...
its nice is a way to know that i am not alone... haha misery loves company
. yeah guess i might have to start lots of DNA. at least, the digestion is rather efficient... In the beginning I was also advised not to use TE buffer, but now I just do it with TE and never had problems, but in theory I think that it can give problems because of the neutralizing effect of the EDTA. I know someone here that uses TE, but with a 10x diluted concentration EDTA.
And fortunate a ligation can be done with only a couple of ng DNA
I was also told not to use TE buffer in the first place because it could affect my ligation- but since then we changed kits and the current one only works well with (if possible preheated) TE buffer. I tried it and my ligation efficiency (which is pretty good ) didn't change... maybe it depends on what T4 ligase you use. Ours is from Applychem and seems to be quite indifferent to most changes in conditions
) didn't change... maybe it depends on what T4 ligase you use. Ours is from Applychem and seems to be quite indifferent to most changes in conditions I loaded more digested DNA/well so that I cut out less of the gel during purification. Used 2 instead of 1 column to purify, pooling them together at the elution stage. Eluted with warm water (~45-50degC). 20ng/ul !!
just set up ligation... hope the cells transform tomorrow
I spoke to someone who who does a lot of cloning stuff and he told me that its normal to lose so much. i'm just peeved that Qiagen claims abt 85% recovery but i am not getting that. Using more plasmid, using more enzymes.. sighs.. this just isn't efficient
What you really need to check is the pH of you're elution buffer or MQ! It should be around pH8.
If the pH is too low (pH7 or lower) DNA does not elute from the columns. With the qiagen gelextraction kit i get a recovery around 70% if the pH of my MQ is adjusted to pH8!!
another tip: every batch of MQ has a different pH at our lab, so I have to check it all the time!
good luck!