Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-rATP, T4 polynucleotide kinase, and buffer (3). After incubation at 37degC, reactions are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The gel percentage and electrophoresis conditions varied depending on the sizes of the DNA molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing.

Protocol

1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed:

		sterile ddH2O				q.s 
		10X kinase buffer			1 ul
		DNA							x ul
		[[gamma]]-[32-P]-rATP		10 uCi
		T4 polynucleotide kinase	1 ul (3U/ul)
									10 ul

2. Incubate at 37degC for 30-60 minutes.

3. Heat the reaction at 65degC for 10 minutes to inactivate the kinase.

G. Bacterial cell maintenance

Four strains of E. coli are used in these studies: JM101 for M13 infection and isolation (4), XL1BMRF' (Stratagene) for M13 or pUC-based DNA transformation (5), and ED8767 for cosmid DNA transformation (6-8). To maintain their respective F' episomes necessary for M13 viral infection (9), JM101 is streaked onto a M9 minimal media (modified from that given in reference (1) plate and XL1BMRF' is streaked onto an LB plate (1) containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid media are inoculated with a smear of several colonies and incubated at 37degC for 8 hours, and those cultures then are transferred into 50 ml of respective liquid media and further incubated 12-16 hours. Glycerol is added to a final concentration of 20%, and the glycerol stock cultures are distributed in 1.3 ml aliquots and frozen at -70degC until use (1).

Protocol

1. Streak a culture of the bacterial cell strain onto an agar plate of the respective medium, listed below, and incubate at 37degC overnight.

 E. coli strain           Agar Medium/Liquid Media
	XL1BMRF' (Stratagene)			LB-Tet
	JM101							M9
	ED8767							LB

3. Transfer the 2 ml culture into an Ehrlenmeyer flask containing 50 ml of the respective liquid media and further incubate overnight (12-16 hours) at 37degC with shaking at 250 rpm.

4. Add 12.5 ml of sterile glycerol for a final concentration of 20%, and distribute the culture in 1.3 ml aliquots into 12 X 75 mm Falcon tubes.

5. Store glycerol cell stocks frozen at -70degC until use.

Notes on Restriction/Modification Bacterial Strains:

1. EcoK (alternate=EcoB)-hsdRMS genes=attack DNA not protected by adenine methylation. (ED8767 is EcoK methylation -). (10)

2. mcrA (modified cytosine restriction), mcrBC, and mrr=methylation requiring systems that attack DNA only when it IS methylated (Ed8767 is mrr+, so methylated adenines will be restricted. Clone can carry methylation activity.) (10)

3. In general, it is best to use a strain lacking Mcr and Mrr systems when cloning genomic DNA from an organism with methylcytosine such as mammals, higher plants , and many prokaryotes.(11)

4. The use of D(mrr-hsd-mcrB) hosts=general methylation tolerance and suitability for clones with N6 methyladenine as well as 5mC (as with bacterial DNAs). (12)

5. XL1-Blue MRF'=D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F' proAB, lacIqZDM15, Tn10(tetr)].

Host Mutation Descriptions:

ara	Inability to utilize arabinose.
deoR	Regulatory gene that allows for constitutive synthesis for genes involved in 
deoxyribose synthesis.  Allows for the uptake of large plasmids. 
endA	DNA specific endonuclease I.  Mutation shown to improve yield and quality of DNA 
from plasmid minipreps.
F'	F' episome, male E. coli host.  Necessary for M13 infection.
galK	Inability to utilize galactose.
galT	Inability to utilize galactose.
gyrA	Mutation in DNA gyrase.  Confers resistance to nalidixic acid.
hfl	High frequency of lysogeny.  Mutation increases lambda lysogeny by inactivating specific 
protease.
lacI 	Repressor protein of lac  operon.  LacIq  is a mutant lacI that overproduces the 
repressor protein.
lacY	Lactose utilization; galactosidase permease (M protein).
lacZ	b-D-galactosidase; lactose utilization.  Cells with lacZ mutations produce white 
colonies in the  presence of X-gal;  wild type produce blue colonies.
lacZdM15	A specific N-terminal deletion which permits the a-complementation segment present 
on a phagemid or plasmid vector to make functional lacZ  protein.
Dlon	Deletion of the lon  protease.  Reduces degradation of b-galactosidase fusion proteins 
to enhance antibody screening of l libraries.
malA	Inability to utilize maltose.
proAB	Mutants require proline for growth in minimal media.
recA	Gene central to general recombination and DNA repair.  Mutation eliminates general 
recombination and renders bacteria sensitive to UV light.
rec BCD	Exonuclease V. Mutation in recB or recC reduces general recombination to a hundredth 
of its normal level and affects DNA repair.
relA	Relaxed phenotype; permits RNA synthesis in the absence of protein synthesis.
rspL	30S ribosomal sub-unit protein S12.  Mutation makes cells resistant to streptomycin.  
Also written strA. 
recJ	Exonuclease involved in alternate recombination pathways of E. coli.
strA	See rspL.
sbcBC	Exonuclease I.  Permits general recombination in recBC mutants.
supE	Supressor of amber (UAG) mutations.  Some phage require a mutation in this gene in order 
to grow.
supF	Supressor of amber (UAG) mutations.  Some phage require a mutation in this gene in order 
to grow.
thi-1	Mutants require vitamin B1(thiamine) for growth on minimal media.
traD36	mutation inactivates conjugal transfer of F' episome.
umuC	Component of SOS repair pathway.
uvrC	Component  of UV excision pathway.
xylA	Inability to utilize xylose.
dam	DNA adenine methylase/ Mutation blocks methylation of Adenine residues in the recognition 
sequence 5'-G*ATC-3' (*=methylated)
dcm	DNA cytosine methylase/Mutation blocks methylation of cytosine residues in the recognition 
sequences 5'-C*CAGG-3' or 5'-C*CTGG-3' (*=methylated)
hsdM	E. coli methylase/ Mutation blocks sequence specific methylation AN6*ACNNNNNNGTGC or 
GCN6*ACNNNNNNGTT (*=methylated).  DNA isloated from a HsdM- strain will be restricted by a HsdR+ 
host.
hsd R17	Restriction negative and modification positive.
(rK-, mK+) Allows cloning of DNA without cleavage by endogenous restriction endonucleases.  DNA 
prepared from hosts with this marker can efficiently transform rK+ E. coli  hosts.
hsdS20	Restriction negative and modification negative.
(rB-, mB-)	Allows cloning of DNA without cleavage by endogenous restriction endonucleases .  DNA 
prepared from hosts with this marker is unmethylated by the hsdS20 modificationsystem.
mcrA	E. coli  restriction system/ Mutation prevents McrA restriction of methylated DNA of 
sequence 5'-C*CGG (*=methylated).
mcrCB	E. coli  restriction system/ Mutation prevents McrCB restriction of methylated DNA of 
sequence 5'-G5*C, 5'-G5h*C, or 5'-GN4*C (*=methylated).
mrr	E. coli  restriction system/ Mutation prevents Mrr restriction of methylated DNA of sequence 
5'-G*AC or 5'-C*AG (*=methylated). Mutation also prevents McrF restriction of methylated cytosine 
sequences.

cmr	Chloramphenicol resistance
kanr	Kanamycin resistance
tetr	Tetracycline resistance
strr	Streptomycin resistance
D	Indicates a deletion of genes following it.
Tn10	A transposon that normally codes for tetr
Tn5	A transposon that normally codes for kanr
spi-	Refers to red-gam- mutant derivatives of lambda defined by their ability to form 
plaques on E. coli  P2 lysogens.
Commonly used bacterial strains

C600	- F-, e14, mcrA, thr-1 supE44, thi-1, leuB6, lacY1, tonA21, l-
	-for plating lambda (gt10) libraries, grows well in L broth, 2x TY, plate on NZYDT+Mg.
	-Huynh, Young, and Davis (1985) DNA Cloning, Vol. 1, 56-110.
DH1	- F-, recA1, endA1, gyrA96, thi-1, hsdR17 (rk-, mk+), supE44, relA1, l-
	-for plasmid transformation, grows well on L broth and plates.
	-Hanahan (1983) J. Mol. Biol. 166, 557-580.
XL1Blue-MRF' - D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, 
lac, l-, [F'proAB, lac IqZDM15, Tn10 (tetr)]  -For plating or glycerol  stocks, grow in LB 
with 20 mg/ml of tetracycline.  For transfection, grow in tryptone broth containing 10 mM 
MgSO4 and 0.2% maltose. (No antibiotic--Mg++ interferes with tetracycline action.)  For picking 
plaques, grow glycerol stock in LB to an O.D. of 0.5 at 600 nm (2.5 hours?). When at 0.5, add 
MgSO4 to a final concentration of 10 mM.
SURE Cells - Stratagene - e14(mcrA), D(mcrCB- hsdSMR-mrr)171, sbcC, recB, recJ, umuC::Tn5 
(kanr), uvrC,  supE44, lac, gyrA96, relA1, thi-1, end A1[F'proAB, lacIqDM15, Tn10(tetr)]. 
An uncharacterized mutation enhances the a - complementation to give a more intense blue color 
on plates containing X-gal and IPTG.
GM272 - F-, hsdR544 (rk-, mk-), supE44, supF58, lacY1 or 苐acIZY6, galK2, galT22, metB1m, trpR55,
l-
	-for plasmid transformation, grows well in 2x TY, TYE, L broth and plates.
	-Hanahan (1983) J. Mol. Biol. 166, 557-580.
HB101	- F-, hsdS20 (rb-, mb-), supE44, ara14, galK2, lacY1, proA2, rpsL20 (strR), xyl-5, mtl-1,  
l-, recA13, mcrA(+), mcrB(-)
	-for plasmid transformation, grows well in 2x TY, TYE, L broth and plates.
	-Raleigh and Wilson (1986) Proc. Natl. Acad. Sci. USA  83, 9070-9074.
JM101 - supE, thi, ?lac-proAB), [F', traD36, proAB, lacIqZ芃15], restriction: (rk+, mk+), mcrA+
	-for M13 transformation, grow on minimal medium to maintain F episome, grows  well in 2x TY,
	plate on TY or lambda agar.
	-Yanisch-Perron et al. (1985) Gene  33, 103-119.
XL-1 blue	recA1, endA1, gyrA96, thi, hsdR17 (rk+, mk+), supE44, relA1,  l-, lac, [F', proAB, 
lacIqZ芃15, Tn10 (tetR)]
	-for M13 and plasmid transformation, grow in 2x TY + 10 礸/ml Tet, plate on TY  agar + 10 礸/ml 
	Tet (Tet maintains F episome).
	-Bullock, et al. (1987) BioTechniques  5, 376-379.
GM2929 - from B. Bachman, Yale E.coli Genetic Stock Center (CSGC#7080); M.Marinus strain; sex F-;
(ara-14, leuB6, fhuA13, lacY1, tsx-78, supE44, [glnV44], galK2, galT22, l-, mcrA, dcm-6, hisG4,[Oc],
 rfbD1, rpsL136, dam-13::Tn9, xyl-5, mtl-1, recF143, thi-1, mcrB, hsdR2.)
MC1000 - (araD139, D[ara-leu]7679, galU, galK, D[lac]174, rpsL, thi-1).  obtained from the McCarthy
lab at the University of Oklahoma.
ED8767 (F-,e14-[mcrA],supE44,supF58,hsdS3[rB-mB-], recA56, galK2,galT22,metB1, lac-3 or lac3Y1 - 
obtained from Nora Heisterkamp and used as the host for abl and bcr cosmids.