Standard Operating Procedure
Title:Southern Hybridiztion Dept:Agronomy Lab: Agronomy B411, B406, G313 Supervisor:Randy Shoemaker
Probe preparation (on ice):
1. Thaw out on ice:
a. OLB
b. BSA
c. lambda
2. Label tubes.
3. add 28 uL of H2O to each probe tube and lambda's tube.
4. add appropriate probe to each probe's tube
a. use between 30 to 40 ng of probe per tube.
5. add 2 uL of lambda to its tube.
6. heat at 100oC fro 5 minutes, then ice.
7. make cocktail (for 4 probes, mix up enough for 5).
a. 50 uL OLB
b. 10 uL BSA
c. 2 uL H2O
8. place Klenow tube in stratacooler.
9. move to hot room.
10. add 25 uL 32P dCTP to cocktail (5 uL per probe)
11. add 3 uL Klenow to cocktail (0.5 uL Klenow per probe).
12. add 18 uL cocktail to each probe's tube and lambda's tube.
13. allow to react for 4 hours.
Hybridization of membranes:
distilled H2O
55
110
20X SSC
30
60
20% SDS
5
10
1.0 M NaPO4
2.5
Mix, heat for 30 seconds per 100 ml in microwave.
50X
6.5
13
10 mg/ml Herring sperm DNA*
1.0
2
* denature for 10 minutes at 100oC, then ice for 3 minutes.
Mix, with stirring magnet, solution.
Soak membranes in 5X SSC for 20 minutes before hybridizing.
Roll-up membranes and place in bottles.
Add 25 ml hybridization solution per bottle/probe.
Procedure 3
Checking incorporation of probes:
1. cut out a small piece of Chromatography paper 6 cm long.
2. on rough side draw a line, in pencil, 1 cm from bottom.
3. mark 5 dots on line (4 probes and lambda) 0.5 cm apart and number.
4. place 0.5 uL of each probe on a dot.
a. correspond dot and probe.
b. last dot is for lambda, always.
5. cover bottom of beaker with 0.7 M sodium phosphate (pH 3.5).
6. put blotted paper in beaker and cover with saran wrap.
a. dots at the bottom.
7. leave for 20 minutes (don't let it reach top of paper).
8. allow to air dry for 10 minutes.
9. wrap in saran wrap.
10. place between 2 Plexiglass sheets.
11. take to darkroom.
12. cut out small piece of film from scrap box.
a. second drawer in right corner.
13. place over paper, between Plexiglass.
a. notch corner to orient after development.
14. cover for 3 minutes.
15. place in developer for 4 minutes (use clothespins).
16. place in 1% acetic acid for 1 minute.
17. place in fixer for 4 minutes then in H2O bath for 10 minutes.
Procedure 4
Adding probes to bottles:
1. add 10 uL labeled lambda to 1 ml H2O.
2. add 10 uL (lambda and H2O) to 1 ml H2O.
3. add 10 uL (lambda + H2O + H2O) to each probe's tube.
4. heat probes for 5 minutes at 100oC.
5. briefly spin down tubes.
6. ice tubes for 3 minutes.
7. add 58 uL labeled probes to bottles.
a. keep tubes on ice.
8. put bottles in carousel for 14 hours.
Procedure 5
Washing membranes:
1. prepare 2 2-liter solutions.
a. 50 ml 20X SSC
b. 50 ml 20% SDS
c. 1900 ml ddH2O
2. heat each 2 liter solution for 10 minutes, on high (microwave)
a. place in 65oC incubator until needed.
3. pour 2 liter into a sandwich box.
4. remove lids and pour excess into waste brown jug.
5. using long forceps, remove membranes from bottle.
8. place membranes in wash solution.
9. after removing all membranes, transfer to second wash solution.
10. cover box and place in 60oC water bath for 30 minutes.
11. dispose of initial wash solution into brown jug.
12. prepare another 2 liter wash solution with same ingredients.
13. transfer membranes to a second box containing new wash solution.
14. cover box and place in 60oC H2O bath for 30 minutes.
15. after second wash, check radioactivity.
a. if below 500 counts, blot dry and wrap in saran wrap.
b. if above 500 counts, perform a third 30 minute wash.
- use new solution with same ingredients.
16. load film in darkroom and place in a plastic bag.
17. place i -70oC freezer for duration of exposure.
a. if below 300 counts, expose for 4 to 5 days.
b. if above 300 counts, expose for 2 to 3 days.
