question:problem during the plasmid isolation - (Apr/11/2007 )
i'm a student of microbiolgy
i had done the plasmid and DNA isolation but i faced several problems.
thus i would like to know the major problem and the solution
-why do we use phenol,ethanol,and isoamil alcohol in the miniprep of plasmid
-i can't see any band of plasmid in my gel!
-how can we get high purity of plasmid isolation?is there any tips.
maybe you could fall back on one ofe the commercially available miniprep-kits. good yield, high purity...
In plasmid isolation phenol helps in removal of proteins( remember to caliberate it's ph to 7.5 by TE buffer), ethanol wash is given so as to remove the extra salts from isolated DNA & Isoamyl alc reduces the foaming when the aq phase is extracted with Chloroform.
well as far as the absence of band is concerned i think u r actually some crucial step.....due to which u r not getting plasmid.....it may be the very last step too.....isopropanol precipitation....I used to get a tiny visible pellet after this step.....
Thanks Poonam for your info about Isoamyl alcohol. I didnot have this knowledge.
phenol drive protein precipitation. Chloroform helps phase separation (more hydrophobic than phenol) and IAA is for chloroform stabilisation and participates also at phase separation.
ethanol is for removing salts.
isopropanol should be mixed 1:1 V/V with aqueous upper phase to be efficent, invert tube and spin.
for high quality plasmid preparation, i would advice to go for a kit. better to remove endotoxins.
If ur lysis and pptation steps are working fine (one can make out while one is doing these) then the problem could be that u donot dry the ethanol after washing . Even if a litl bit is left the DNA will not dissolve.
Maybe you accidentally lose the pellet during the isopropanol step. If you add 1µl of Glycogen you can make the pellet visible thus making it easier to keep it in the tube. Besides that take a small aliqout after all steps after the chloroform isolation and put in on a gel. This way you could identify the crucial step you are doing wrong.
Are you doing Alkaline lysis?
If you get a little amount of DNA, check the first stage on the protocol, try to resuspend very well the pellet, this is a crucial step, becouse if you have more cells in suspension, in the second step you lysate more cells.
For the phenol step you should after start, dilute your sample in 300 microlitres or more, becouse if you lose some volume by pipeting you loose less DNA than working in low volumes.