short protocols in molecular biology...... - maybe too short??? (Jun/17/2006 )
does somebody also have this book??? anyway miniprep of plasmid takes usually whole day with me....now we've got this book and it says no phenol/chloroform extraction, no RNase (add if necessary), no PEG precipitation just incubate in three solutions and that is it! 
...im soooooo tempted to try it.... 
...do you think such plasmid can't be used for cloning....?? 
If there is no column purification or some sort of purification step such as the phenol/chloro extraction, your prep is probably pretty dirty. I don't know if the DNA would be that useful for anything more than a qualitative test for determining if you have plasmid or maybe pcr. But you can do pcr straight from a colony or lysate...
I guess you are talking about the protocol in which they suggest to use sol1, sol2 and sol3 and then to add Sosium acetate and EtOH to precipitate the DNA. Well, if you don't want to digest the plasmid you obtain (or if you want to digest it with EcoRI), it works. The DNA will be very full of salt and therefore very difficult to digest. This is my experience. I like a lot the fast protocols but this is not very good...
ok i got it....but what about if I purify with phenol/chloroform, ethanol precipitate but no RNase and PEG precipitation...I want to do restriction enzyme digestion and ligation after that....will it be ok?
phenol/chloroform will clean up most of the junk, EtOH will get rid of the worst of the salt, so it should be fine for digestion.
BTW, it sounds like plasmid preps from the bad old days (or good) before columns took over, and we had to do things for ourselves. When was the book published?
for minipreps and restriction, i do plasmid purif without phenol chloroform, and don't add RNase. It's ok for screening.
But i don't do trust it for a cell tranformation for ex...
Can you not use a spin column kit? We use Promega's Wizard kit, coupled with a vacuum manifold, and I can do a dozen or so minipreps in like twenty minutes. The DNA is pure enough for everything -- we even sequence right from it.
This can not be a cost issue -- compare the cost savings of your time (going from all day to less than half an hour), the savings and safety associated by not having to deal with organic solvents like phenol and chloroform, the consistent results obtained each time, etc. and you save waaayyyy more money than the cost of the kit.
yes Promega's Wizard Kit is really effective and resonable.
...and all the protocols are like that...purification from the gel has no phenol/chloroform after it too... so since im transforming after that i guess that means i have to do it all the way including PEG precipitation...
as i understand it my lab is very very poor and traditional methods are so established noone wants to try anything new....that is just so common here to spend a whole day for miniprep .
Do the math for them. Calculate the cost per prep of doing it your way versus the cost per prep of doing it the newer way. Include employee costs at a constant rate in both calculations, to account for the time differences. Include costs associated with storage and/or disposal of hazardous solvents. Include the cost of media -- spin-column minipreps take a three to five ml overnight culture -- what does your method need?
There's no easy way to quantify the success rate of one protocol over another until you try it and compare it to your old method. How often do you have to do a miniprep over because of unsatisfactory results using your current method? I can tell you from long-time (ten years?) use of the Wizard kit (and similar spin-column based kits) that my failure rate due to recovering DNA of insufficient quality or quantity (because of a failure of the kit, not a property of the plasmid) approaches zero quite closely.
Get together with your local sales rep, explain the situation. He or she will probably be willing to help you with this type of economic argument. He or she will also likely be willing to provide you with a sample kit and perhaps a price break if it is clear that you're trying to change the culture of the lab and introduce his/her consumable products...
I can't imagine taking a whole day for a miniprep... 