Sickle Cell & Southern

Blot

By :Grant Akalonu
CHE 447-01
Objective/Introduction

The purpose of this experiment was to understand how southern blotting is used to
diagnose sickle cell anemia. Southern blotting is a technique used in molecular biology to detect
specific DNA sequences in a DNA sample. In this experiment, we studied a hypothetical
situation in which two parents are tested to see if the possess the sickle cell trait.

Methods/Materials

50X TAE electrophoresis buffer was diluted to 1X.

0.8% agarose gel was prepared by mixing 0.23 g of UltraSpec agarose powder in 30mL
of 1X TAE electrophoresis buffer. The mixture was heated in a microwave and then
cooled to 60C before being poured into the bed with the comb sitting firmly and evenly
across the bed.
The gel was allowed to solidify completely. Then it was placed in the electrophoresis
chamber and 18-20 L of each DNA sample was placed into separate wells.
The gel ran for 1 hour at 135V.
After electrophoresis, the agarose gel was depurinated in 200 mL 0.25M HCl at room
temperature for 8 minutes.
The HCl was discarded and the gel was rinsed several times with 200 mL of distilled
water.
The gel was soaked in 200 mL of denaturation solution (0.5M NaOH/0.6M HCl) for 15
minutes on a shaker. The denaturation solution was discarded and soaked with another
200 mL of denaturation solution. The new denaturation solution was saved to a nylon
membrane.
The gel was removed for the tray and placed face down on plastic wrap a on a flat level
surface.
The nylon membrane was trimmed to the size of the gel, soaked withdenaturation
solution for 5 minutes, and then placed on top of the inverted gel.
Blotting filter paper was trimmed to the same size as the membrane and placed ontop of
the membrane.
A test tube was rolled across the beaker to remove bubbles. Then a stack of paper towels
was placed on the fliltr paper with a 400 mL beaker on top of it for 1 hour. The gel was
wrapped carefully and then saved in the fridge overnight.
After the paper towels were removed, the gel was removed from the membrane and air
dried.
The membrane was placed in a clean tray with the DNA side up, and soaked in 100 mL
of Blue-Blot solution for 15 minutes.
The Blue-Blot solution was discarded, and rinsed with 200 mL of water until the
membrane was destained.
Results

Lane Tube Sample

1 A Sickle Cell
Gene Sample
2 B Sickle Cell
Carrier
3 C Normal Gene
4 D Mother DNA
Sample
5 E Child
DNA
Sample
6 F Father
Fig.1. Membrane Result. From left to right, lanes 1-6. DNA Sample

Discussion & Conclusion

The results of southern blotting are shown in Figure 1. At the top of the gel, the negative
current form electrophoresis pushes the samples toward the positive end of the gel (the bottom).
The rate which the samples move down the gel depends on the size of the DNA fragments. The
larger DNA fragments move slower down the gel and remain at the top, and the smaller ones
move quicker and move closer to the bottom of the gel. Based on the result in Figure 1, it
appears that the parents are both carriers of the sickle cell gene, and the child is normal. The
travel distance of the fragments for both parents match that of a carrier, and the travel distance of
the child matches that of a normal individual.