Plasmid isolation from bioluminescence bacteria - Alkaline lysis (Nov/17/2008 )

this is my first post in forum ...

recently i am doing my final year project ... i tried several times to isolate plasmid DNA from bioluminescence bacteria using alkaline lysis method ... but still cant get any plasmid until now ...

here are the steps i do:

get overnight culture of bacteria (i always culture bacteria for 18 hours, but without shaking, is it the main reason for my failure? because my groupmates who are doing genomic DNA extraction successfully isolate gDNA without shaking ...)

centrifuge to get bacteria pellet (i suspect my desired plasmid is low-copy plasmid, so i increase the volume of broth up to 3 ml instead of 1.5 ml ...)

resuspend in 100ul of GET buffer (my stock buffer volume is 50ml, and since glucose cannot be autoclaved, i used membrane filter to discard all the contaminant ... is my way correct?)

add 100ul of 0.4N NaOH/1% SDS and incubate in ice for 5 min (the alkaline solution is freshly prepared just before the addition)

add 150ul of 5M potassium acetate and incubate in ice for 5 min

centrifuge to get supernatant

add 1 volume of isopropanol and incubate in ice for 20 min

centrifuge to get pellet

wash with 1ml ethanol

air dry the pellet


after i do all these steps, i found white or even transparent pellet in my microfuge tubes ... and after i run gel, what i get is the band above 10kb or even larger than gDNA ... what have i done wrong? i really have no clue ... and desperate because due date for final year project is approaching ...

really hope someone can give me suggestion ... thanks ...

That might just be your plasmid. . . . .
don't look at your friend genomic dna as reference..

we all know genomic dna is huge, but as u can see in the process of purifying it it got sheared and cut thus reducing it size. furthermore the column used to isolate the dna got a size restriction as to the maximum size of dna that could be trapped and eluted out.

Hi luciferase,
Shaking is necessary to aerate the bacteria, so they will grow (this is what I have found): I use 225 RPM.
What percentage of ethanol do you use to wash the pellet? I find that 70% is good.
mito1

Are you sure you have a plasmid at all in the wild type strain? What organism is this? Perhaps you have no plasmid present, and are purifying and analyzing genomic DNA.

All the replies above could be right.

What you saw might be your plasmid and you could try digest it with a few restriction enzymes (assuming you don't know the sequence of the plasmid). If lucky you could hit a couple sites and linearize or fragment the plasmid and if its gDNA it will be a smear.

The difference of plasmid yield could be several folds with vs without shaking.

Alkaline lysis is very dirty and you have tons of RNA smear that might mask your plasmid band and the proteins might trap RNA or DNA in the well and you may see this >10kb staff. We are offering free AquaPlasmid sample now, if you want to give it a shot.

Can you post a picture of your gel?