Southern blotting is a method which is used to:
a) Identify specific sequences of DNA in which DNA fragments are separated by electrophoresis, transferred to a membrane, and identified with a suitable probe.
By this way, DNA is transferred from an agarose gel to a membrane, where the DNA can be subsequently probed for a specific sequence.
b) Detects restriction fragments following a restriction enzyme digest of genomic DNA (RFLPs).
c) Detects RT-PCR (Reverse transcriptase-PCR) fragments.
d) Can be used to screen cDNA libraries.
Key
to a successful transfer
a) No
air bubblee
--> Roll out with a pipet
b) No short circuits
--> Use parafilm or old xray film to create a “mask”
c) Plenty of paper towels
d) Time
--> Good to set up just before you leave and let it transfer overnight (or at least 6 hours)
Hybridization means the fragment contains DNA is complementary to the probe. The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods.
