Mystery band after Phenol:Chloro:IAA Extraction after restriction digest - (Apr/21/2008 )
I have a plasmid that I am going to use for In vitro transcription. I am linearlizing with EcoRI from NEB and following Ambion's recomendations for the preparation of template DNA. After EcoRI digest, I stop the reaction with EDTA and Protinase K treatment, I extract the DNA with Phenol:Chloroform:IAA. When I run an aliquot of the recovered DNA on a gel, I see 2 bands (~4kb and ~2kb). If I run some of the digest that was not Prot K or PCI'd, I only see 1 band (4kb).
Im very perplexed and was wondering if anyone has any experience or insight with this phenomenon!
i should clarify that the expected size of linear plasmid is 4kb.
I would heat kill EcoRI rather than use EDTA and Prot K. You should be able to make good DNA by following the heat kill with a column purification (the enzyme cleanup from Qiagen, for example). How long are you digesting DNA? What buffer are you using? EcoRI can show star activity with the wrong buffer or high glycerol concentrations (don't add more than 10% enzyme to a reaction, I try to always make it less than 5%). It's also best to keep DNA concentrations relatively low, less than 2 ug in a 100 ul reaction.
Well I dont think its any issues with star activity or other problems with the enzyme since I dont see any problems if i run an aliquot before Prot K and PCI forllowed by EtOH ppt. But to answer your questions, I digest 20ug in 100 ul for 2 hours in EcoRI Buffer (unique) with 2 ul of enzyme... then add another 1 ul of EcoRI for 1 hour.
What I think I am going to do is do a column purify the product and just try a transcription reaction with what I get from that.
Hopefully that will work, but Im still curious to find out if other people have seen this happen before!