P1 DNA preparation

P1 DNA preparation

bionet.genome.arabidopsis Nov 21 1995

We have published a paper describing an Arabidopsis P1 genomic library (Liu et. al., 1995. Plant J. 351-358), and the library is available from the Ohio Stock Center. In addition to the P1 plasmid replicon (single-copy ), the P1 vector (pAd10-sacBII, Pierce et. al. 1992. PNAS, pp2056-2060) also contains a P1 lytic replicon (multi-copy) under the control of the LacZ promoter/operator so that the copy number of the P1 clones can be amplified to get a high yield of DNA. However, recently we found that the copy number of the clones actually cannot be amplified by IPTG-induction, even at concentrations of 1-10 mM IPTG. On the contrary, the DNA yields from cultures with IPTG are slightly lower (~20%) than those without IPTG, due to the poor growth of cells in the presence of IPTG. The reason for the lack of induction is unclear. Despite failure to amplify the copy number, a moderate amount (0.5-1ug/ml culture) of the clone DNA is still easy to get from a mini- or midi-preparation by the alkali method (DNA yields were determined by fluorescence with Hoechst 33258 dye, which is not affected by RNA contamination, and the low level of E. coli DNA contamination was confirmed by agarose gel electrophoresis). Dr. Pierce (1992) described in "Methods In Enzymology" (Wu R., ed., pp.549-574) that the yield of P1 clone DNA from 10 ml LB culture containing 1 mM IPTG is 1-2 ug. Since this yield is even lower than those I obtained from cultures with or without IPTG, it appears he did not achieve induction either. I compared the DNA yields between our P1 clones and a mini-F-based (single copy) BAC clone (120 kb) and found they were about the same. All the same, if you use the following protocol, you can easily isolate enough DNA from your target P1 clones:
  1. (day-one morning). Streak out P1 clone cells onto an LB agar plate containing 25 ug/ml of kanamycin, culture at 37 C for 20-24 h.

  2. (day-two morning). Inoculate a single colony into 5-10 ml (or more) of LB containing 20 ug/ml kanamycin (without IPTG). Note that direct inoculation with glycerol cell stock often results in lower yields of DNA, thus is not recommended.

  3. Culture at 37 C for 10-14 h to reach saturation. Over culture (longer than 15 h) is not recommended. If preparation is not done at once, store the cells at 4 C overnight (even longer is OK). This storage at 4 C is advantageous for reducing amount of RNA in the cells.

  4. (day-three). Set a microfuge at 4 C. Collect cells of 3 ml (or 4 ml) of culture in each 1.5-ml (or 2-ml) tube(s) by repeating two centrifuging at 8000 rpm for 2 minutes. remove any medium with a pipet tip.

  5. Resuspend the cells in 100 (or 130) ul solution I (about 30-35 ul solution I for every 1 ml culture) by vortexing. Add 10 ul of lysozyme (10 mg/ml in 10 mM Tris.Cl pH 8.0) and mix.
    * Solutions I, II and III are prepared as described in "Molecular Cloning" (pp. 1.25-1.26). It is better to chill solution III at -20 C before use.

  6. Add 200 (or 260) ul freshly prepared solution II. Mix by GENTLY inverting the tubes 10 times. Store the tubes on ice or at room temperature for 5-6 min but no longer.
    * In this and all the following steps, VORTEXING MUST BE AVOIDED. Vortexing may shear the high molecular weight clone DNA and host chromosome DNA, and increase host chromosome DNA contamination.

  7. Add 150 (or 200) ul solution III. Mix by gently inverting the tubes 5 times. Store the tubes on ice for 3-5 minutes.

  8. Invert the tubes 3 times, then centrifuge at top speed for 5 min at 4 centi- digree. At the end of centrifuging, set the microfuge at 15 C.

  9. Transfer the supernatant to fresh tubes. Add 0.8-1 vol. phenol/chloroform and mix by gently inverting. Centriguge at top speed for 5 min.
    * This phenol/chloroform extraction step should not be omitted, which helps reduce contamination of the host chromosome DNA and impurities.

  10. Add 2 vol. ethanol at room temperature, mix by inverting, stand for 2 min, and centrifuge at 12000-15000 rpm for 8-5 minutes. The pellet is washed extensively with 70% ethanol. This is done by poking the pellet (submerged in 1 ml 70% ethanol) with the pipet tip and swishing it around to achieve thorough rinsing. Centifuge as above.

  11. Without over-drying the pellet , the DNA is redissolve in 10 ul TE per ml culture containing 20 ug/ml RNase A by heating at ~50 C and gentle mixing. Now you should have DNA in concentrations 50-100 ng/ul. Store the DNA at 4 C.

  12. The DNA can be used directly for various enzyme manipulations, or purified again by phenol/chloroform extraction and ethanol precipitation, or by spot-dialysis on a VSWP02500 filter (Millipore) floating on 1/2 or 1/3 TE for 30-60 minutes (during the dialysis the DNA volume increases, and you can concentrate the DNA by spotting the solution on to a Saran wrap laid over a heat-block (40-50 C) untill the volume reduces to 1/2-1/3.
    * If the whole clone DNA is labeled as probe by random-primed method, shear the DNA by vortexing, sonication, .ect, before heat-denaturation.


Y.-G. Liu
Mitsui Plant Biotechnology Research Institute
E.mail: tsu00131@koryu.statci.go.jp