Consistent Lambda preps, Thanks

Hong Dang hdang at cns.neusc.bcm.tmc.edu
Wed Jul 28 21:46:13 EST 1993


I post a request for the above subject, and thanks to Antje Haase and Andre Hamel for
they wonderful suggestions. Here are the info in case other people are interested.

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when I was doing  a lot of lambda preps I found that it is most
important to have a very well lysed culture in the first place. It's got to
be almost transparent with protein flakes floating everywhere. If you don't
get that it is not worth starting a DNA prep at all. You may set up several
cultures in parallel using various amounts of cells with a constant amount of
phage. Phage taken from fresh plaques work best! 
The other important thing is aeration, make sure you have a big anough flask
for the culture you are growing. Often the yield is higher from a smaller
culture: 50ml culture (in 500 ml flask) gives you up to 1mg of lambda DNA. 

DNA prep itself is straightforward, basically following the "standard method
for purification of Bacteriophage lambda" in "Maniatis" up to the PEG
precipitation and centrifugation. It needs about 50
ug DNAse and RNAse for the initial step though (not 1ug). Don't do the CsCl
gradient but simply resuspend the pellet in SM/20mM EDTA/0.5% SDS, add
50ug/ml proteinase K, incubate 1hr 65C, phenolize, EtOH precipitate. For best
quality DNA don't spin but "fish out" your DNA with a pipette tip.
Always works very well and gives high quality DNA! 

Good luck

Antje  



______________________________________________________________________________
Antje Haase
Menzies School of Health Research
Casuarina, NT 0811
Australia
_______________________________________________________________________________
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Within following methods is what we use for Lambda preps. We always get
good preps following to the letter.

If ANY questions, please drop a note;

cheers, Andre
                            ********************
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
Infectious & Genetic Disease                     FAX: (204) 945-8062     
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
                            ********************
						
        Here's a few methods (it was less work for me to send as one larger
file rather than sending just the ssDNA prep'n method). See also recent
Biotechniques (Vol.14, No.4, April 1993), there's an article with simpler,
at times better ssDNA extraction method (pg 540), there's also an article on
ExoIII pretreatement of ssDNA PRIOR to use for mutagenesis ... increases %
mutants made (pg 544). Also better plasmid prep method on pg 532
(optimized PEG ppt'n in order to minimize RNA contamination).


In general (unless otherwise stated), perform ALL ext'ns on ice, and
microfuging done at top speed at 4oC.

TE is 10 mM Tris, pH 8.0, 1 mM Na2EDTA, pH 8.0

all phenol used for DNAs is buffered by saturating with 0.3 vol. 0.1 M
Tris, pH 7 to 8.

all chloroform contains either 2% (final v/v) either n-butanol or isoamyl
alcohol
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                 Making Lambda gt11 Phage Stocks

1/   Start with log phase recipient cells (eg Y1090, Y1091).

2/   Mix 0.25 ml of cells with 50 ul of appropriate dilution of
     phage suspension (either from a single plaque or from plates
     of plaques). 

3/   For lambda gt11 incubate at 42oC for 15 min.
4/   Add 3 ml of molten (45oC) top agarose, mix well and pour
     onto LA plate containing Mg++, pH 7.8. 

5/   Incubate at 42oC for between 6 and 12 hrs.

6/   Flood plates containing confluent plaques with 5 ml of SM
     (suspension medium);
     50 mM Tris-HCl, pH 7.5
     100 mM NaCl
     10 mM MgSO4
     0.01 % gelatin 
     Sterilize SM by autoclaving.

7/   Incubate at RT for 2 hrs with gentle swirling. Scrape off top
     agarose, vortex for 1 minute then centrifuge for 15 min. at
     10- 20 krpm. Add CHCl3 to 0.3% final v/v and storage this
     supernatant at 4oC.

8/   Determine titre; generally, phage titre ought to be about
     10 E10 pfu/ml.

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                  Lambda Phage DNA Preparation

     Modified from Kaslow (1986) Nucleic Acids Research 14:6767

1)   Inoculate 1L of LB pH 7.8, 10 mM MgCl2 (no maltose) with 20
     mL of overnight host E.coli that was grown in same type of
     LB but did contain 0.2% maltose. Do not supplement the 1 L
     culture with maltose or phage will release their DNA into
     lysed cells in addition to live cells, decreasing the yield of DNA.
2)   Incubate at 37oC (42oC is better) at about 200 rpm rotation
     until OD650 is about 0.5. 
3)   Add phage extracted from confluent plates at dose of about 2
     plates worth of phage per 100 mL of culture. (5 mL of SM per
     plate, top agar scraped into centrifuge tube, sit r 4 hrs,  
     spin 10 krpm/20 min). This gives an m.o.i. of about 1.
4)   Incubate until clearing occurs (plus stringy cell debris),  
     usually takes about 2- 3 hrs.
5)   To lysed culture add;
          NaCl to 1 Molar final concentration (6 gm/100mL)
          2% CHCl3 (final vol./vol.)
          1 ug/mL (final conc.) DNase
          1 ug/mL (final conc.) RNase A, RNase T1 mixture or RNase 1 alone.
6)   Continue incubation with shaking for another hour.
7)   Let stand about 10 min. and collect aqueous phase (pipet out
     CHCl3 from bottom.
8)   Centrifuge in vinyl or glass centrifuge bottles (clear
     polycarbonate tubes will be destroyed by residual CHCl3) at
     6 krpm for about 20 min.
9)   Decant supernatant. Iff clear add solid PEG 8000 to 12% w:v.
     If not clear filter through Whatman GFA glass fiber filters
     prior to addition of PEG. 
10)  After PEG is dissolved let stand only 1 hour at 4oC. At this
     stage an overnight precipitation is okay but 1 hr is better
     because less cellular DNA and RNA will ppt with the phage.
11)  Centrifuge PEG ppt at 5000 krpm for 15 min. (7 krpm for 10
     min.) Make certain to mark the tube where the pellet is!
12)  Carefully decant supernatant (save in case "pellet" is dislodged 
     while draining and resuspend the "pellet" (actually is a loose film) 
     in 6 mL TES/Mg [10 mM Tris HCl pH 7- 8, 1 mM EDTA, 20 mM NaCl and 
     20 mM Mg (Cl or S04)].
13)  To resuspended phage add to final conc. 50 ug/mL DNase and
     100 ug/mL RNase A, gently swirl about 20 times, then incubate r 2 hrs. 
     at 37oC (overnight/weekend is better).
14)  Add Proteinase K to 100 ug/mL and incubate at 55oC.
15)  After 10 min. add SDS to 0.5% final w:v.
16)  After 20 min. further add Na2EDTA to 20 mM.
17)  Following 10 min. further incubation perform two phenol (Tris buffered, 
     pH 7- 8), three phenol:chloroform:butanol (50:50:1) extractions or until
     interface is clear, then final two chloroform:butanol (50:1) extractions. 
     Vortex vigorously for 1 minute for each extraction.
18)  Ethanol:NH4OAc (2.5 M final conc. ammonium aceteate, 2-3 times volume 
     ethanol) ppt. in glass Corex centrifuge tubes, vortex well, sit at
     rm. temp. about 20 min. then centrifuge at 8 krpm for 20 min. Drain, 
     discard supernatant. Quick microfuge, carefulle remove/discard remaining 
     supernatant.No need to vacuum dry at this point.

     FROM THIS POINT ONWARD, VORTEX GENTLY as phage DNA may SHEAR.

19)  Dissolve pellet in 1- 2 mL of TE at 65oC. Add NaCl to 0.4 M
     To this add an equal vol. of 13% w:v PEG 8000. Vortex well.
20)  Let stand on ice for 1 hr. (Overnight is too long because small RNA
     degradation products will precipitate otherwise).
21)  Centrifuge at 8 krpm at 4oC for 30 min. Drain/discard supernatant.
22)  Dissolve pellet in 1- 2 mL TE.
23)  Extract twice with 3 vol. of CHCl3 (needed to remove PEG).
24)  Ethanol:NaOAC ppt at -20oC for 1 hr., centrifuge, wash pellet in -20oC
     80% ethanol by adding same volume 80% ethanol as was used in 
     precipitation, vortex, centrifuge again, drain/discard supernatant 
     again. Wash pellet again but this time in 100% ethanol, vortex, spin, 
     drain/discard supernatant, vacuum dry.

25)  Dissolve pellet in 1 mL TE at 65oC.

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