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I have to isolate a large plasmid from yeast and transform it in E. coli. After transformation, I often get no colonies. One reason for that is the yeast mini prep hasn't worked or the DNA concentration is too low. I tried two different isolation protocols:

  • Zymoprep I yeast plasmid prep kit

    From the doc: "based on the old E. coli alkaline lysis method with our Zymolyase added in the first solution."

  • Phenol chloroform YAC isolation protocol

    used by Dan Gibson et. al] for their huge constructs (>100kb)

    They use an expensive Qiagen large-construct column at the end, in order to purify and visualize the yeast extract on a gel, but I skip that step because the Qiagen column cost $40 per yeast clone which doesn't scale if we do many of these.

How can I troubleshoot and optimize the above-mentioned protocols? Are there any other protocols that might be more suitable for a large plasmid and don't use too expensive materials?

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2 Answers 2

A dependable protocol for yeast DNA extraction I used to use was broadly similar to the protocols you cite but included an ethanol precipitation before and after the P:C extractions. The only expensive material was time. The general outline was:

  1. Alkaline lysis
  2. Ethanol precipitation
  3. RNase A treatment of resuspended DNA for 15 minutes
  4. Phenol:chloroform extraction (x2)
  5. Ethanol precipitation

You probably know that the ethanol precipitation lore is that the yield is better the longer and colder you do your ethanol precipitations. I only ever did reverse transcriptase reactions a few times, but the template DNA I prepared spent the night in ethanol at -80C to maximize yield. Another place to double-check is that your yeast cultures are sufficiently dense before starting.

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What should be the OD of the culture? What is the volume of the culture? –  Gergana Vandova Mar 14 '12 at 18:51
    
I took 1.5mL of a "dense overnight culture" for this protocol. I don't remember being particularly careful about the OD for this (and the OD is relatively strain-dependent anyway). For us, "overnight" meant the culture was in a healthy stationary phase and that was easily gauged by eye. If you need to get a feel for the growth properties of your strain, start a culture in the morning and take periodic OD600 readings throughout the day. That should give you a clear picture of when you'll have the maximum number of healthy cells. –  yamad Mar 16 '12 at 10:39

I've successfully used the Zymoprep kit, but only for smaller plasmids in 2-hybrid experiments. So their cell lysis enzyme/buffer system works well, but I'd guess that your 42kb plasmid is precipitating out with the genomic DNA.

The standard Qiagen midi columns are capable of capturing large constructs. This has worked wonders for me when I was purifying large BACmids from E. coli - and gets the cost down to $10/sample (it does require additional buffer, but that's a minor expense). It might be worth trying the Qiagen yeast DNA protocols for the midi kit and see if you can isolate your large plasmid?

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And for what it's worth, the technical reps for Qiagen were very helpful on the phone. Unlike Invitrogen. I dread every call to Invitrogen technical service. >:[ –  Amy Feb 21 '12 at 18:37

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