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
[[gamma]]-[32-P]-rATP (35020) ICN and T4 polynucleotide kinase (70031) from United States Biochemicals.
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
2. Pick several colonies into a 12 X 75 mm Falcon tube containing a 2 ml aliquot of the respective liquid media, and incubate for 8-10 hours at 37degC with shaking at 250 rpm.
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)].