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Flashcards in Southern Blotting Deck (21):
1

Principle of Southern Blotting

the transfer of fragmented DNA from an agarose gel to a nylon or nitrocellulose membrane via capillary action

2

Preparation of Genomic DNA

Restriction enzymes are used to cut the DNA into specific fragment lengths (20kb and more)

3

Preparation of tissue and blood cells

Cells are incubated in a buffer containing a detergent such as SDS (sodium dodcyl sulfate) which will disrupt the cell membranes and release high-molecular weight DNA which is then purified and fragmented with restriction enzymes

4

Preparation of bacterial cells

the cell wall of the bacteria is gently broken open using a treatment of Lyzosyme and ethylenediaminetetraacetate (EDTA) which is then purified and fragmented using restriction enzymes.

5

Electrophoresis

The fragmented DNA is then sorted by size. Samples are place within wells on the gel and a current is run through the buffer. The positive charge pulls the DNA through the gel (with the smaller fragments moving faster) Dye mixed with the buffer allows for the visualization of the bands.

6

Gel pre-treatment: step 1

gel is soaked in 0.25molL-1 HCl for 30min to break individual brands into even smaller fragments for easier transfer to the membrane.

7

Gel pre-treatment 2

An alkaline solution is then used to break the hydrogen bonds between DNA strands creating single strands. Aiding the transfer and binding to the membrane, as well as ensuring the base pairs are available for hybridization with a probe. If a nitrocellulose membrane is used the gel is then neutralized in a Tris-salt buffer as DNA will not bind at a pH greater than 9.0

8

Blotting - step 1

The gel is placed on a filter paper wick that forms a connection between the gel and the reservoir of high-salt buffer (20xSSC - saline-sodium citrate)

9

Blotting - step 2

A membrane (originally nitrocellulose) is placed on top of the gel and covered with a tower of paper towels that are held in place with a weight.

10

Blotting - step 3

Capillary action results in the buffer soaking through the filter paper wick and into the paper towels. As the buffer passes through the gel, the DNA fragments are carried to the membrane where they become bound. For fragments up to 15kb in length this can take approximately 18 hours.

11

Alternative to capillary action

Vacuum blotting - where pressure is used to draw the buffer through the gel and membrane more rapidly - reducing the transfer time to 30mins.

12

4 advantages of nylon membrane over nitrocellulose

1) less fragile - is not damaged by handled and can be re-hybridized up to ten times.
2) under a positively charged membrane with alkaline transfer buffer the transferred DNA becomes covalently bonded reducing the possible loss of DNA through leeching.
3) transfer time is reduced to 2 hours (from 18)
4) can bind DNA fragments at 50bp in length (nitrocellulose are effective with fragments longer than 500bp)

13

Fixing the DNA to the membrane

Nitrocellulose: baked at 80'C for 2 hours forming non-covalent but semi-permanent attachment.
Nylon: exposed to UV radiation forming covalent bonds.

14

Pre-hybridization

the membrane is soaked in a solution designed to block unused DNA binding sites to prevent nonspecific DNA binding of the probe. This can take between 15min and 3 hours at 68'C depending on the type of membrane.

15

Pre-hybridization solution

Contains non-biological polymeric compounds such as polyvinylpyrrolidone and/or biological polymers such as bovine serum albumin (BSA), dried milk or DNA from an unrelated organism (salmon sperm is a popular choice)

16

Hybridization

Carried out in a high-salt buffer containing a detergent: 2xSSC + 1% SDS.

17

Increasing specificity

must be carried out at a stringency that results in the specific probe-target hybrid remaining stable while all other hybrids are unstable.

18

Washing

Carried out after hybridization, this is done in order to remove non-hybridized probe

19

What is Tm

This is the highest temperature at which the hybrid is stable.


For most 15-25mer oligonucleotides is between 40 and 90'C and is calculated by:
(2 x AT nucleotides) + (4 x GC nucleotides) 'C

20

What is increased stringency determined by

This is determined by
1) the composition of the hybridization buffer (hybrid stablity is dependant on ionic strength with destabilizing agents such as formamide disrupting hydrogen bonds of unstable hybrids)
2) the temperature (a fully base paired hybrid has a higher melting temperature than one that is only partially hybridized) at which it is carried out.

21

What are the 2 critical points of hybridization?

1) enough probe DNA must be hybridized to the target restriction fragment to produce a clear signal to the detection method used.
2) the specificity of the reaction. if the specificity is too low the DNA probe will bind to fragments which has a partial complementarity.