Week 2: Agarose Gel Electrophoresis; Restriction Mapping; Southern Transfer

Introduction

Agarose gel electrophoresis separates nucleic acids (DNA and RNA) based on size. When applying an electrical current, negatively charged nucleic acids in solution migrate through an agarose gel toward its positive end, with smaller fragments migrating faster (See Module 1 Week 2 for details). Meanwhile, the uncut plasmid DNA may appear in one or more of the following conformations, and each conformation runs at different speeds during agarose gel electrophoresis. Different plasmid conformations listed in the order of electrophoretic mobility (slowest to fastest from (i) to (v)) are: (i) Nicked Open-Circular DNA, which has one strand broken; (ii) Relaxed Circular DNA, which is enzymatically relaxed and fully intact with both strands uncut; (iii) Linear DNA, which has two free ends because both strands are cut; (v) Supercoiled or Covalently Closed-Circular DNA, which is intact with both strands intact, and a twist built-in, resulting in a compact form. Due to the excessive alkalinity during plasmid isolation, there is sometimes (iv) supercoiled denatured DNA, like supercoiled DNA, but with unpaired regions, making it run as a sharp band faster than the supercoiled DNA.¹³ There may also be (v) single-stranded supercoiled DNA that runs at the front end.

For Southern blot analysis, after restriction digestion and agarose gel electrophoresis, DNAs in the gel are denatured and transferred onto the membrane (Southern transfer). There are different ways to transfer DNA from gels to membranes. Since the capillary transfer methods are easy, it is the most popular system for Southern transfer with agarose gels. The upward capillary transfer of DNA from gel to the nitrocellulose or nylon membrane has been used for decades (Figure 2-4).¹⁴ The challenges of the upward capillary transfer include the gel becoming crushed by the weighted filter paper and stack of paper towels laid on the top, slowing down the blotting process and possibly reducing the amount of DNA transferred. Therefore, a downward capillary method is quicker and can result in a better and complete transfer.¹⁵ There is also the bi-directional capillary method, where two blots are made from one gel.

This lab exercise focuses on agarose gel electrophoresis of the restriction-digested plasmid DNA from the last lab. A DNA band corresponding to the NBS sequence from the pSCA7 (T1-T3-W6) is excised from the gel, which will be extracted and quantified for DIG probe synthesis in the next lab (Week 3). Meanwhile, the rest of the DNA is transferred from the gel onto a membrane by the upward capillary method (Figure 2-4).

Procedure

A. Agarose Gel Electrophoresis

1. Heat ~0.45 g of agarose in 45 ml of 1 X TAE (0.9 ml of 50 X TAE 44.1 ml of H₂O) in a 250 ml flask until the agarose completely melts.
2. Swirl the mixture and allow the mixture to cool to ~60°C.
3. Add 5 µl GelRed and mix well by swirling, but avoid introducing bubbles.
4. Pour the agarose solution into a pre-sealed gel tray with the sample comb containing 8 wells and allow it to set.
5. After gelling, place the gel tray in the electrophoresis box and add ~270 ml 1 X TAE to cover the gel.
6. Load 5 µl of molecular weight DNA marker into lane 5 followed by loading the entire volume (24 µl including 6x loading buffer) of each restriction-digested DNA sample to its corresponding lane, as shown below.
   

   Lane	1	2	3	4	5	6	7
   Sample	Uncut	EcoRI	PstI	Both	DNA marker	X	NBS insert

7. Electrophorese at ~100 V until the bromophenol blue is at least ~3/4 through the gel.
8. While waiting, weigh a 1.5 ml microfuge tube for step 10. Record the weight.
9. Visualize the gel containing DNA fragments under the Biospectrum UVP GelDoc-It®2 Imager and photograph with a fluorescent ruler alongside the lanes.
10. With a clean, sharp scalpel, cut the band containing the known NBS DNA fragment in lane 7 from the gel, put it in the pre-weighed microfuge tube prepared in step 8, weigh it again, and store it in the freezer for next week’s DNA extraction and DIG-labeling. Gel excision is conducted with a portable UV box and requires a UV protective shield or a pair of glasses/goggles for protection.

B. Southern Transfer

1. Use a razor blade or scalpel to trim off lanes 7 (gel excision) and 8 (unused) and a ruler to measure the size of the rest of gel.
2. Transfer the gel to a small container.
3. Add a proper volume of denaturing solution (1 M NaCl, 0.5 M NaOH) to cover the gel (~70 ml) and shake at low speed for 30 min.
4. While waiting, use a paper cutter to cut a stack of unfolded paper towels ( > 1.5 inches thick after being pressed down) that matches the size of the gel measured in step 1.
5. Cut a piece of nylon membrane and 2 pieces of Whatman paper the same size as the gel and place them in a dish of distilled H₂O, followed by 2XSSC solution. Wear gloves when you handle both!
6. Cut a piece of Whatman paper the same length but twice the width as the gel tray to be a wick.
7. Start the Southern blot assembly by flipping your gel tray over and placing it in a 2^nd container (Figure 2-4).
8. Place the Whatman paper from step 6 on top and add a proper amount of denaturing solution (1 M NaCl, 0.5 M NaOH) to the paper (~200 ml).
9. Pour off the denaturing solution from the gel (step 3) and transfer the gel upside down on the Whatman paper.
10. Place the nylon membrane from step 5 on top of the gel. Carefully lay down the membrane, and do not move it once it touches the gel. Smooth out any bubbles with a glass tube or rod.
11. Place the pre-wet Whatman papers one by one on top of the membrane and remove any bubbles again.
12. Cut pieces of plastic wrap and lay them along the edge of the gel, extending to cover any opening of the container and prevent the transfer buffer from evaporating overnight.
13. Place the stack of paper towels from step 4 on top of the Whatman paper.
14. Place a small weight (e.g., a pipette box) on the whole stack and allow the DNA to transfer overnight.
15. Next morning, remove the paper towels but leave the Whatman paper, membrane, and gel together. Note: wear gloves for the following steps.
16. Flip the entire stack right-side-up and mark the position of each well on the membrane by poking through the gel piece of each well with a pencil. This is to keep track of the DNA side of the membrane and the starting point of electrophoresis. After you flip the stack, the gel is on the top (right-side-up) with the membrane underneath, and the Whatman paper is at the bottom.
17. Remove the gel and filter papers from the membrane. Rinse the membrane in 2XSSC.
18. Place the membrane with the DNA side face up on sheets of Kimwipe.
19. Air-dry the membrane.
20. Place a few more Kimwipes on the membrane and then wrap them loosely in aluminum foil.
21. Label your section and group number on the aluminum foil and store it in the refrigerator.

Data Analysis and Discussion

1. What is the % of agarose in the gel used in this lab?
2. Make a calibration curve and estimate the sizes of all the bands in your restriction digest lanes 2–4.
3. Construct a restriction map of plasmid based on the results from question 2.