Take the 2-minute tour ×
Biology Stack Exchange is a question and answer site for biology researchers, academics, and students. It's 100% free, no registration required.

I'm trying to find a good protocol for plasmid minipreps and I'm looking at 3 preps I've found:

  1. Using phenol/chloroform
    • extract with phenol:chloroform:isoamylalcohol,
    • isopropanol precipitation, 12,000g spin down,
    • rinse with cold 70% ethanol.
  2. Using lysozyme
    • lyse with lysozyme,
    • remove pellet,
    • isopropanol precipitation,
    • wash with cold 80% ethanol.
  3. Using alkaline lysis
    • open cells with 80% glucose in EDTA buffer,
    • add SDS and NaOH,
    • pellet protein/membrane with acetic acid/acetate,
    • ispropanol precipitation,
    • wash with cold 70% ethanol.

They all differ in how to break open the cells and separate plasmids from the rest of the cell -- quite a bit. Can anyone help me figure out which protocol is best here?

share|improve this question
7  
Are you sure you want to have students use phenol? Most labs probably use kits for DNA extraction (e.g. Quiagen MiniPrep). It's much easier and also safer. –  Mad Scientist Dec 14 '11 at 22:44
    
i'm going to mark Reven's answer as the best since it helps me most choose amongst the protocols. but thanks for pointing out how cheap the qiagen kits are- i'd thought they were much worse. you do learn something by using a protocol, but dang the columns are easy! –  shigeta Dec 15 '11 at 20:53
    
FYI while its true about $1-2 per pre for spin columns, but the reagent cost for SDS/NaOH is about 40 cents per prep. you have to do over 100 preps to see the savings though. –  shigeta Dec 15 '11 at 21:22
    
Just wanted to update that i bought columns from biobasic and while it is not as for instruction as these basic protocols, they are certainly cheap enough and should be considered for the classroom - upvotes all around. –  shigeta Oct 19 '12 at 18:16
add comment

5 Answers

up vote 6 down vote accepted

In my experience, the P:C:I method will get you higher yields, and is a bit more simple (in steps and chems involved), but as @Mad Scientist has said, phenol use might be an issue. It depends on the age of your students.

share|improve this answer
    
It is not only the age of the students, it also depends on the available facilities. You DO need a hood for instance, and you need to have a service available for the disposal of phenol. –  nico Dec 22 '11 at 10:16
add comment

To save cost, you can buy spin columns alone from suppliers such as Syd Labs (http://www.sydlabs.com/spin-column-for-plasmid-miniprep-p110.htm) and make home-made buffers. The suppliers normally provide the buffer recipe.

share|improve this answer
add comment

I think the spin column kits are the way to go. As mentioned already, the benefits of the kits are that they are easy, safe, and (most importantly) how almost every actual lab does plasmid purification these days.

The biggest criticism of the commercial kits is that you can get by easily without knowing anything about what is actually happening in the tube. However, as noted in the comments to Nick T's answer, there is no secret proprietary technology in the kits--they use the alkaline lysis method. This means that you can still teach the mechanism in the class while getting the benefit of the spin columns.

One option, for teaching purposes, is to make all of your own solutions and just use the spin columns on the supernatant after pelleting the precipitated membrane/protein. That way, you get the benefit of insisting on real names for the solutions (NaOH is a more educational name than P2) and you get to use the spin columns in place of the ethanol precipitation. I believe there are cheap sources that just sell the spin columns, although I have never used them. You want to avoid the ethanol precipitation because it adds a bare minimum of 30 minutes to the protocol (for my protocols, it was more like an 1.5 hours or more). And waiting for ethanol to evaporate so you can resuspend is like watching paint dry unless you have a speed vac.

Yield shouldn't be a big issue in a class situation, but you can boost yield with the column by prolonging the final elution step. I think the given instruction is to let the TE buffer (Tris) "elute" for 1 minute before the final spin, but I have routinely let the TE sit for about 15-30 minutes before spinning down. The difference in yield blew out the exposure on the gel camera.

There are certain cases where phenol:chloroform extractions are still desirable, but this is definitely not one of them. I used to do P:Cs routinely when working with RNA and yeast genomic DNA. I have never seen anyone do anything other than a miniprep when preparing plasmids. What's more, the hassle of working in a fume hood and the risk of phenol burns encouraged a lot of people in the lab to prefer the spin kits even when working with genomic DNA.

share|improve this answer
    
after some effort we did move to the spin kits... –  shigeta Jun 16 '12 at 16:43
add comment

Using spin columns, my professor and I extracted ~30 plasmids from ~30 samples in a few hours. It's easy, cheap, and it yields good quality plasmid. My sample was quantified by UV spec and it was at about 200ng/ul.

share|improve this answer
add comment

The protocol I used in my genetics lab course was alkaline lysis followed by ethanol precipitation similar to this one. Nothing terribly toxic or requiring a fume hood (at least at the small volumes used).

The more practical, reliable method is to use a miniprep kit (usually spin columns) from one of several suppliers (Qiagen, Promega, Bio-Rad, etc.) which are fairly affordable at $1-2 per prep.

share|improve this answer
1  
If I recall correctly, alkaline lysis is the underlying method behind Qiagen DNA purification kits (miniprep, etc). With the spin columns from the kit, you get to cut out the alcohol precipitation and drying. Maybe doesn't sound like much, but it is worth the price of the kit because it eliminates more than 10 minutes and a sensitive step from the protocol. My two cents: you do not want to use phenol in the classroom. –  yamad Dec 15 '11 at 0:10
    
All the prep kits I've used (Qiagen, Promega, Invitrogen) do use alkaline lysis. I like the columns just for the sake of not having to worry about totally evaporating all the ethanol (I use them with a vacuum manifold so I suck for an extra minute or two to let it all evaporate) or looking for some half-invisible pellet. –  Nick T Dec 15 '11 at 0:19
    
this is quite useful - thanks! –  shigeta Dec 20 '11 at 17:57
    
Liking this more once i found the qiagen recipes on Open Wet Ware.. openwetware.org/wiki/Miniprep/Qiagen_kit makes me feel better about using it in a class. –  shigeta Dec 21 '11 at 16:12
add comment

Your Answer

 
discard

By posting your answer, you agree to the privacy policy and terms of service.

Not the answer you're looking for? Browse other questions tagged or ask your own question.