P1 DNA preparation

Y.Ryu tsu01131 at KORYU.STATCI.GO.JP
Tue Nov 21 05:49:52 EST 1995


Dear Netters,

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 centi-digree 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 centi-digree 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 centi-digree overnight (even longer is OK). This storage at 
centi-digree is advantageous for reducing amount of RNA in the cells.

4 (day-three). Set a microfuge at 4 centi-digree. 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 centi-digree 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 centi-digree.

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 centi-digree and 
gentle mixing. Now you should have DNA in concentrations 50-100 ng/ul. 
Store the DNA at 4 centi-digree.

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 centi-digree) 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.

If you have any quetions, please let me know.

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




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