what is your DNA concentration after gel extraction? - (Jun/11/2012 )
I am using Qiagen's gel extraction kit for years now but it only gives me around 10 ng/ul of DNA. I have tried adding Binding Buffer to the column just before washing with PE buffer, but that also doesn't help. I have also used Intron's Megaquick which extractcs a little more than 10 ng/ul.
Is it always like this? I keep losing a lot of DNA, PCR product or digested plasmids. There are also other kits which are not column based, like Qiagen's Qiaex II, but you lose a lot of DNA with them too.
what is your concentration after gel extraction?
This depends, of course, on the amount in your band before extraction. After several years, I completely gave up on gel extraction, partly for this reason, and partly due to DNA damage by UV. There is almost always a better way of doing things that avoid it.
Yeah, I get about the same amount, no matter the method. I find it isn't too bad for cloning PCR'd inserts as you typically only need tiny amounts for the molar ratio thing. However, I generally avoid it if at all possible.
Phage, may I know what method you use if you want to avoid gel extraction?
Bob1, I also had no problem with having 10 ng/ul previously, because as you said we don't need much DNA for cloning and ligation. But now I am struggling with Overlap Extension PCR and I need high amount of template DNA for the second PCR. I cannot add more than 10-15 ul of the purified PCR product to my second PCR, and I am worried if this is the reason my PCR fails.
It's a series of techniques, not just one. The most important is one that switches antibiotic resistance at each cloning stage. The inserts have resistance A and B. The vector backbone is chosen to have resistance C. The backbone is prepared by PCR with low amounts of template, and DpnI digested to remove template which has not been amplified. The cloning is done by ligating the insert from A, the insert from B, and the PCR product backbone without any purification. Enzymes are chosen to be easily heat killed. Select on antibiotic C. We use Kan, Tet, Chloramphenicol. With three, you can always do this reaction.
Assembly of BioBrick standard biological parts using three antibiotic assembly.
Shetty R, Lizarazo M, Rettberg R, Knight TF.
Methods Enzymol. 2011;498:311-26. PMID: 21601683
Thanks Phage, I learned something new today