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SOUTHERN BLOTTING

Outline

Interview with Edwin Southern - Protocols: Southern, 2006 - Southern, 1974

DIGESTION - CONDITIONS - REBASE

TRANSFER - Traditional

FIXATION
Crosslink -NH₂ nylon groups and DNA sugar-phosphate backbone
through 5 minutes irradiation using a 256 nm UV lamp

HYBRIDIZATION PROBE (known and visible)
LABELING
- PNK
- Pol
- Tdt

[another use of PNK and TdT in iscChIC-seq, an alternative to ChIP:
 repair by PNK of 5´-OH ends created by Micrococcal nuclease,
 and creation by TdT of poly(G) terminals to bind oligo-dC adaptors]

HYBRIDIZATION CONCEPT

Example: Discovery of Splicing
(1977, 2016  [Figure] - and 2020 Articles; Nobel Prize 1993 - Sharp Lecture)

The incubation temperature (Τ_i) can be estimated from the following formulas
(from Davis et al., "basic Methods in Moleular Biology"):

Τ_i =T_m - 15°C
T_m = 16.6 log[M] + 0.41 [P_GC] + 81.5 - P_m - B/L - 0.65[P_f]

Where:
[M] is the molar concentration of Na⁺, to a maximum of 0.5 (1 x SSC contains 0.165 Μ Na⁺)
[P_GC] is the percent of G or C bases in the oligonucleotide probe (between 30 and 70)
P_m is the percent of mismatched bases, if known
      (each percent of mismatch will alter the T_m by 1°C on the average)
Β is 675 (for synthetic probes up to 100 bases)
L is the probe length in bases
[P_f] is the percent of formamide in the buffer

HYBRIDIZATION REACTIONS - Oven
WASHES

AUTORADIOGRAPHY - Slides by R. yadu
 
NONRADIOACTIVE METHODS