No visible pellet using alkaline lysis method for miniprep - (Oct/09/2006 )
Hi. I really hope that somebody can help me to solve the problem.
I am using alkaline lysis method to extract plasmid DNA from Top10 competent cell. I am using the protocol from 'Short Protocol in Molecular Biology". I started with 1.5ml of broth culture. At the final stage, after adding 95% EtOH and spin for 5 min at room temp, I expect to see some pellet but I couldn't. I thought that my product was so clean that the pellet was invisible. However after adding in 30microL of TE buffer and load 3microL of the plasmid onto 1% agarose gel, the supercoil plasmid band was very faint. My question are:
1. do I expect a pellet to be visible?
2. is it a necessity to incubate the solution after adding in a) GTE, b)NaOH/SDS, and c)potassium acetate?
3. what are the critical steps in alkaline lysis method?
4. what is the ideal plasmid band pattern in agarose gel?
5. i did 30 plasmid prep in one batch. What are the precaution of preparing large amount of plasmid in one goal?
Thank you.
I am using alkaline lysis method to extract plasmid DNA from Top10 competent cell. I am using the protocol from 'Short Protocol in Molecular Biology". I started with 1.5ml of broth culture. At the final stage, after adding 95% EtOH and spin for 5 min at room temp, I expect to see some pellet but I couldn't. I thought that my product was so clean that the pellet was invisible. However after adding in 30microL of TE buffer and load 3microL of the plasmid onto 1% agarose gel, the supercoil plasmid band was very faint. My question are:
1. do I expect a pellet to be visible?
Yes. The pellet should be visible to your eye. It isn't big, but it has presence.
a) No (strong vortexing)
Very much NO (invert mixed solution 5 times)c) no (light vortexing)
Vortexing! Vortexing! and maybe don't hang around more then 2 minutes at the NaOH/SDS alkaline lysis step. Makes lots of bad denatures plasmid DNA if you wait too long at the alkaline lysis step.
Yes, the most important step is vortexing the cell pellet.
1 -Spin down the cell from it growth medium
2- Remove supernatant by aspiration. Keeping cell pellet
3- (This is quite important) You vortex the pellet. You must break up the pellet here, it is very much more difficult anywhere else in the protocol. Once the pellet is broken up
4- Add 100ul GTE solution + RNAse, and the "cell pellet" should go into solution.
5- Add 200ul NaOH/SDS
6- Invert tube 5 time
7- 150ul KAc
How about using isopropanol for the percipitation step instead? At this point your tube contains 400ul of stuff. Adding EtOH will push the volume very close if not right over the limits of the 1.5ml micro centrifuge tube.
I would also spin for 10mins. Maybe it makes a difference, maybe not
Surely it must be the correct band pattern
Well for raw DNA, a strong thick band of supercoiled DNA, very little linear or nicked DNA. And certainly no denatured DNA (whose presence becomes known when a single RE digest is conducted)
Do you mean doing more then 30 minipreps or do you mean doing 100ml midiprep and up (like 10 litre megapreps?)
For more the 30 minipreps... make sure you have good labelling. And be careful when using the adding the alcohols as spillage will remove your labelling.
For larger preps,
-always make sure the pellet is broken up, before adding the lysis solution
-it become rather expensive to use RNAse on megapreps... so use LiCl percipitation to remove the RNA.
As for your problem....
-are you certain you have enough cells... how long is the incubation time?
-is the plamid of interest a high copy plasmid?
see dude, u dont start getting huge yields of plasmids from ur miniprep from the start.
takes alot of time to optimize.
Its good that u r getting atleast a band. I used to lose my plasmid frequently during my minipreps.
Just give it time and perseverance. try as many differnt protocols as u can.
or you can go by the "Karate Kid", "chinese kung fu movie", and DragonBallZ way...
My supervisor started me doing minipreps.... lots and lots of minipreps... 72 colony minipreps, twice a week for a month.... under such a regime... you become very good very fast... able to miniprep with the speed and accuracy of an inhuman robot...
or die
....from having to repeat the experiment.
Thank you guys..I really appreciate it
You must also take into consideration the copy number of your plasmid. Is it a low copy number plasmid? How big is it? How is your starting culture grown? How much of it are you extracting from?
There are several tweaks regarding good yield recovery of low copy number plasmids...
In general, I agree with what's been said. Absolutely no vortexing at the lysis step (I never vortex at ANY step) and no extended incubation in alkaline conditions.