isolation of P1 dnas..help?

R.F.Whittier tsu01135 at KORYU.STATCI.GO.JP
Mon Aug 25 23:35:29 EST 1997


We posted this to the Arabidopsis newsgroup over a year ago, but
since the question of how to isolate P1 clone DNA has surfaced in
this newsgroup, we'll re-post it here. Yao-Guang Liu is now back in
China.

Robert F. Whittier, Ph.D.
Senior Researcher
Mitsui Plant Biotechnology Research Institute
Tsukuba, Japan

E-mail: tsu01135 at koryu.statci.go.jp

*************************************
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, pp 2056-2060) also contains a P1 lytic replicon (multi-
copy) under the control of the lacZ promoter/operator to enable
amplification of plasmid copy number for DNA isolation. However,
recently we found that the copy number does not respond to IPTG,
even at concentrations of 1-10 mM. In fact, IPTG is counter-productive,
lowering DNA yields by about 20%, due to the poor growth
of cells when it is present. To test whether the lytic replicon
might be constitutively active, several clone DNAs were re-introduced 
into E. coli XL1-Blue (lacIq) and DH10B (lacI) strains. We
compared the DNA yields among preparations from the original strain
NS 3529 (lacIq) and other strains, together with that of a mini-F-
based (single copy) BAC clone (100 kb), and found them to be
approximately equal. Thus the lytic replicon appears to be simply
non-functional. The reason is unclear. Pierce and Sternberg (1992)
reported the yield of P1 clone DNA from a 10 ml LB culture
containing 1 mM IPTG to be 1-2 ug (Methods In Enzymology, Wu R.,
ed., Vol. 216, pp. 549-574). Since this yield is even lower than
what we obtained with or without IPTG, it appears they did not
achieve induction either. Even though the plasmid copy number cannot
be amplified, it is still easy to get a moderate amount (0.5-1 ug
DNA/ml culture) of clone DNA from a mini- or midi-preparation by the
alkaline method. This yield estimate is reliable because DNA concentrations were determined by fluorescence with Hoechst 33258
dye, a method not affected by RNA contamination. Using agarose
pulse-field gel electrophoresis, we confirmed that the bulk of the
DNA present was comprised of P1 clone DNA rather than of E. coli
chromosomal contamination. The following protocol is for mini-
preparation from 3 ml of culture, and DNA isolations can be scaled
up proportionately.

1.	(Day one). Streak out P1 clone-bearing cells onto an LB agar
plate containing 25 ug/ml of kanamycin, culture at 37 ¡C for 20-
24 h.

2.	(Day two). Inoculate a single colony into 4 ml (or more) of LB
containing 25 ug/ml kanamycin (without IPTG). Cultures can be
inoculated directly from glycerol cell stocks, but this often
results in lower DNA yields. 


3.	Culture at 37 C for 12-15 h, allowing the culture to reach
saturation. Longer culturing is not recommended. However, the
cells may be stored at 4 C for up to several days at this point
without noticible reduction in DNA yield or quality.

4. 	(Day three). Set a refridgerated microfuge to 4 C. Harvest 3
ml of culture into one 1.5-ml tube by pelleting cells twice at 
8000 rpm for 1 minute each time. Remove any medium with a micropipet.

5.	Resuspend the cells in 100 ul solution I (about 30-35 ul solution
I for every 1 ml of culture) by vortexing. Add 10 ul of lysozyme
(10 mg/ml in 10 mM Tris-HCl 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 ul of solution II. Mix by GENTLY inverting the tubes 10-
15 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 will shear high molecular weight DNA, not only
fragmenting the clone molecules but increasing the levels of host
chromosomal DNA contamination, as well.

7.	Add 150 ul of solution III. Mix by gently inverting the tubes 10
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 C.
     * At the end of this spin, set the microfuge to 15 C.

9.	Transfer the supernatant to fresh tubes. Add 0.8-1 vol.
phenol/chloroform and mix by gently inverting. Centrifuge at
top speed for 5 min.
     * This phenol/chloroform extraction step should not be
omitted; it helps reduce contamination by the host chromosome
along with other impurities. 

10.	Add 2 vol. ethanol at room temperature, mix by inverting, let
stand for 2 min, and centrifuge at 12000-16000 rpm for 5-10
minutes. Wash the pellet extensively with 70% ethanol.

11.	Avoiding over-drying, redissolve the DNA pellet in TE containing
20 ug/ml RNase A (use 10 ul of TE  per ml of culture)
by heating at ca. 50 C and gently mixing. If the same DNA was
prepared in more than one tube, samples can be pooled at this
point. Store the DNA at 4 C. Although the DNA can be used
directly for various enzymatic manipulations, the following steps
are recommended to increase DNA purity and improve cutting by
more finicky enzymes.

12.	To each sample prepared from 3 ml of culture, add TE to a
final volume of 110 ul or more. Insufficient dilution will lead
to coprecipitation of impurities in the following steps. If
either DNA yield or impurity levels are unusually high, more
dilution is called for. Extract again with phenol/chloroform.

13.	Add 0.01 vol. of 1M MgCl2 and 0.5 vol. of 40% polyethylene
glycol #6000 or #8000 (note that no NaCl is included). Mix well,
then pellet by centrifugation at top speed for 10 minutes at 20
C.

14.	Wash the DNA pellet with 70% ethanol and redissolve in 30 ul
of 0.3 M acetate sodium. Reprecipitate with 2.5 vol. cold ethanol.  

___________________________
This protocol was optimized for P1 DNA isolation by Dr. Yao-Guang
Liu over the course of his research at the Mitsui Plant Biotechnology
Research Institute. Any correspondence should be sent
to Dr. Daisuke Shibata (tsu01136 at koryu.statci.go.jp) or Dr. Robert
F. Whittier (tsu01135 at koryu.statci.go.jp) of the same institute.




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