Can phenol be mixed with QG buffer from Qiagen Kit - (Jun/12/2008 )
Does anyone know if QG buffer (which contains guanidine thiocyanate) can be mixed with phenol, phenol/choroform. I need to do a gel extraction and my product is unfortunately in regular agarose and will not melt with heating at 65 degrees. It does however melt quite well with the QG buffer in the Minielute kit from Qiagen. I can't use the kit however because my PCR product is over 8kb. Any help would be appreciated.
I don't know the answer to your question, but you could treat your agarose with agarase to release the DNA. Likely you could also use a small filter and spin the DNA out of the agarose. Electroelution could also be used, typically done by inserting the gel sample in a dialysis bag and immersing in a gel tray with buffer, and running until the DNA is released from the gel and trapped by the dialysis membrane. Concentrated sodium iodide solutions will also dissolve agar at 50C or so, and would be compatible with a follow on phenol/chloroform extraction.
QIAquick kit can purify dna size up to 10 kb.
If you have PCR product, you can use QIAquick pcr purification kit rather than gel extraction. Just a suggestion.
You can use this qiaex II Kit that can purify upto 50Kb fragment from gels:
All great suggestions...thanks everyone. I have the Qiagen PCR purification kit but didn't think I could use it for gels. That seems like the easiest method to use so I will try it. Again...thanks phage 434, dcch and cellcounter for the help.
You are welcome:
PCR purification is great, you also do not expose your DNA to UV and agarose contamination, but beware!
If you are using this 8Kb fragment for cloning, smaller non-specific amplifications (that you can not see in the gel, but are always there) will be preferntially cloned, especially because 8Kb fragment faces more difficulty in getting cloned. So, you will end up screening a lot of non-specific clones.
For most other purposes, and for smaller fragments for cloning, qia PCR purification is the best choice.
Thanks cellcounter....I am actually self ligating the digested fragment (it has a restriction site) and transfecting into Ecoli. I did try the PCR purification kit but found that the PBI buffer that comes with it doesn't allow the agarose to melt when incubated at 65 degrees. I then removed that buffer and added the QG buffer from the gel extraction kit and the gel melted well with that. I then processed the liquid through the Qiagen PCR purification column per the usual protocol. I don't know what I have yet because I didn't get a chance to check the Nanodrop but will keep you updated. Thanks
If you go through and compare protocol between those 2 kits, only the 1st reagent(and some centrifugation steps) makes the difference. So you mentioned you use Buffer QG instead Buffer PB, actually you were doing gel extraction protocol
PCR purification kit should only be used with sample in "liquid" form, meaning a digested or pcr product. Melting gel with buffer PB is a no-no.