Southern blot动物生物技术系

Southern印迹是将 DNA片断从电泳凝胶上直接转移至膜支持物（如硝酸纤维素膜、尼龙膜）上，使 DNA片断固定的技术。

SouthernSouthern 印迹印迹

先将 DNA经限制性内切酶消化成一系列片段，进行琼脂糖凝胶电泳，各片段因分子量不同而彼此分开，然后经碱处理凝胶，使 DNA的片段被变性、中和并通过毛细作用在高盐缓冲液中在原位将单链核酸转印到硝酸纤维膜上，烘干、固定。

分子生物学中使用的标记方法

杂交研究中的核酸探针杂交研究中的核酸探针

核酸杂交探针使用的标记物核酸杂交探针使用的标记物

随机引物的探针标记

DN

A

的限制性酶切

Southern 印记

DNA分子

限制片段限制性酶切割琼脂糖电泳

转移至硝酸纤维素膜上

与放射性标记DNA探针杂交

放射自显影

带有 DNA片段的凝胶

凝胶滤膜

用缓冲液转移DNA

吸附有 DNA片段的膜

Southern印迹法

Albert Lasker Award for Clinical Medical Research

Alec John Jeffreys and Edwin M. SouthernAlec John Jeffreys and Edwin M. Southern for development of two powerful technologies - Southern hybridization and DNA fingerprinting - that together revolutionized human genetics and forensic diagnostics.

Alec John JeffreysUniversity of Leicester (UK)

Edwin SouthernUniversity of Oxford (UK)

Southern Southern 印迹法的历史背景印迹法的历史背景

Southern Hybridization

• An agarose gel is prepared and run according to the standard procedures, then it is stained and photographed to have a record of all the DNA fragments.

• The gel is prepared by first denaturing, then neutralizing, the DNA fragments in the gel

Southern hybridization has many steps and usually requires more than one day (or lab period) to complete. This procedure can be broken down into several sections

Preparing the gel

• After neutralization, the gel is carefully measured. Once the gel dimensions are known, we can begin to assemble the materials needed for Southern transfer.

• The nylon membrane is cut to the size of the gel. The membrane itself is sandwiched between two sheets of protective paper. However, wear gloves when working with the membrane to further protect it from oil and dirt. 

• Two pieces of 3 mm filter paper are also cut to the size of the gel. Students should also prepare the stack of paper towels that are placed on top of the membrane. These should be roughly the same size as the gel

• Cut parafilm strips that are longer than the width and length dimensions of the gel. These are used to "mask" the gel and direct the capillary action through the nylon membrane. Finally, two large 3 mm filter paper wicks are cut for the next step; assembling the buffer tank and wick.

• First, pour buffer into the large pan. Then, carefully wet the wicks. Once the 3 mm filter paper is wet it can tear easily. 

• Next, invert the small pan and place into the larger pan. Finally, carefully center the wicks over the small pan. Make sure there is enough wick on each side to reach into the buffer well.

Assembling the buffer tank and wickA wicking system is assembled that will draw 10x SSC buffer from a well in a large pan up to the gel. The gel will sit on the wicks on top of an "island" made by inverting a smaller pan.

• Carefully tuck the wick into the buffer wells.

• At this point, the gel has been denatured and neutralized and all the materials have been cut to size.  The next step is placing the gel on the filter paper wick.

• Once the gel has been denatured and neutralized, it is ready for Southern transfer.

Placing the gel on the Southern apparatusand preparing the nylon membrane.

• Once the gel has been denatured and neutralized, it is ready for Southern transfer.  The gel is carefully inverted.  When running a gel, the DNA is closer to the bottom. By inverting the gel, the DNA has less far to move through the gel when being transferred to the nylon membrane

The gel is inverted so the wells are now facing down, towards the filter paper wicks

The gel is centered on the filter paper wick, and any air bubbles are gently pushed out

Meanwhile, the nylon membrane is prepared by wetting it in distilled water.

The membrane is separated from the protective paper

Once the membrane is wet, it is ready to place on the gel.

Assembling the Southern apparatus

The nylon membrane is the first item to be placed on the gel.

It is critical to line up the membrane and the gel. Once the membrane is on the gel, transfer starts almost immediately.

Lay down the membrane in a smooth motion, either from the top down or from the center going out.  Then smooth out any air bubbles that may be trapped between the membrane and the gel

The next step is applying the parafilm "mask" around the perimeter of the membrane.  Cover enough of the membrane so the parafilm stays in place, but not enough to block any DNA lanes

Once the parafilm has sealed the nylon membrane, the two filter paper wicks are centered on top of the membrane.  Finally, a stack of paper towels is put on top of the filter paper to encourage the capillary action

The finished Southern apparatus and the results

The completed Southern transfer apparatus.  The DNA will transfer overnight. The membrane is removed, washed, and baked at 70'C. The final procedure is developing the nylon membrane using nonradioactive probe detection.

The Southern gel, showing all the DNA bands from cleaving Lambda with different restriction enzymes. Since we know the sequence that is complimentary to our probe, we should be able to determine which fragment will be detected by the probe

The Southern blot shows only one band in each lane. This band corresponds to the band on the gel which contains the Lambda sequence that is complimentary to our probe. This sequence occurs only once in each digest and hence there is only one band per lane

1. 动物组织 (2mg) 用液氮研磨后 , 加入 500ul lysis buffer , 振荡至彻底悬浮 .2. 加入 20ul 蛋白酶 K 溶液 (20mg/ml), 混匀 .55℃ 放置 4-16 小时 , 直到组织溶解 .3. 加入等体积的酚：氯仿：异戊醇（ 25 ： 24 ： 1 ） , 充分颠倒混匀 .4. 14000rpm 离心 5min ，上清转移置另一干净离心管中5. 重复抽提一次6. 加 2 倍体积无水乙醇颠倒混合沉淀 DNA 。7. 14000rpm 离心 10min ， DNA 沉淀形成白色絮状物，去上清。 8. 70% 乙醇洗沉淀两次，室温干燥9. 加入 50-100ul TE ， 55 3-4℃ 小时溶解

DNA 提取操作步骤