Southern blot

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					                                  Southern blot


1. wash gel for 15min in freshly made 0.25M HCl (for large fragment)
2. remove HCl and wash with DDW.
3. Wash twice in denaturation solution (15min each)
4. Wash twice in neutralization solution (15 min each)
5. Wet membrane in 2X SSC and transfer O.N in 10X SSC.
6. Wash membrane briefly in 6X SSC and wipe remains of agarose.
7. Crosslink (twice @1200)
8. Wet membrane in 6X SSC (leave for 10 min @ R.T)
9.  Remove wash and put in prehybridization solution for several hours @ 65 0 C.
10. replace prehybridization solution with the same solution to which 1  106 cpm/ml
   of radiolabeled probe was added. Incubate O.N @ 650 C
                 Do not forget to boil the probe!!!
11. wash twice with 6X SSC to remove excess probe.
12. Wash for 20 min in 1X SSC, 0.1% SDS @ 650 C.
13. Wash for 30 min in 0.5X SSC, 0.1% SDS @ 650 C
14. Wash for 30 min in 0.1X SSC, 0.1% SDS @ 650 C
    Check with Geiger between washes to know when to stop.


     Denaturation solution:             20x SSC
     NaCl - 1.5M                         350 gr. NaCl
     NaOH-0.5M                           176.4 gr. trisodium citrate
                                          H2O to 2L
     Neutralization solution:             prehybridization;
     NaCl - 1.5M                           6X SSC
     Tris pH 7.2 - 1M                      10X Denhart
     EDTA - 1mM                            0.1% SDS
                                            0.1% NaPPi
                                    100g/ml salmon sperm DNA(boil before use)

				
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