PCR, Southern Blot

[Zzmed]

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Guys,

under PCR in FA, it says that after denaturation, annealing, elongation we do an agarose gel electrophoresis to seperate the PCR fragments we made.

This agarose gel electophoresis thing is Southern blot, isn't it??

 

[withrye]

Not usually. After PCR, you typically just wash the gel with ethidium bromide, which intercalates all the DNA and fluoresces under UV light. This will let you see everything that was amplified.

In Southern blotting, the DNA is transferred to blotting paper (hence the name) and the blotting paper is washed in a hybrid probe specific to a particular sequence, and later you visualize everything the probe hybridized with.

With PCR, you hope to gain specificity with your initial primer. With Southern blotting, you take a whole mess of DNA and later probe it with a specific complementary strand to see if it was there.

 

[TheOwl007]

step 1: do PCR (consists of denaturation, annealing, elongation)

step 2: take the sample that underwent PCR and perform agarose gel electrophoresis to seperate the PCR fragments based on size

step 3:
option 1: stain the gel with ethidium bromide to visualize every single band of that the DNA that was seperated ino
option 2: southern blot (denature the DNA in the gel so its single stranded --> place filter paper/nitorcellulose on the gel so it will absorb the DNA --> take the paper/nitro and incubate with a probe [probe=small piece radioactive/labelled DNA] --> image the nitro to detect where the probe anneals. This is the band (or bands) that contains your sequence of interest.)

Summary: the southern allows you to find a particular band of interest. The Ethidium bromiide stain will light up every band in the gel.