Materials and reagents

Biological materials

1. Fetal bovine serum Premium (Bio-Techne, catalog number: S11150)

2. Oligonucleotides (Integrated DNA technologies or other relevant source)

   1. Complementary sense and antisense strands

   2. Options to add modified bases including 2'-O-methyl and 2'-fluoro nucleotides, modifications to the backbone, including phosphorothioate backbone modification, replacing phosphodiester bonds in the oligo backbone at specific locations, or other modifications.

1. Nuclease-free water (Invitrogen, catalog number: AM9932)

2. Tris base, molecular biology grade (Millipore Sigma, catalog number: 648310)

3. Concentrated hydrochloric acid (HCl) (Fisher Chemical, catalog number: SA49)

4. Sodium chloride (NaCl) (Millipore Sigma, catalog number: S9888)

5. EDTA (0.5 M), pH 8.0, RNase-free (Invitrogen, catalog number: AM9260G)

6. Taurine (Millipore Sigma, catalog number: T0625)

7. Acrylamide/Bis solution 29:1 (30%) (Bio-Rad, catalog number: 1610156)

8. Ammonium persulfate (APS) (Millipore Sigma, catalog number: A3678)

9. TEMED (tetramethylethylenediamine) (Bio-Rad, catalog number: 1610800)

10. 2× RNA loading dye (Thermo Scientific, catalog number: R0641)

11. GelRed nucleic acid gel stain (Fisher Scientific, Biotium, catalog number: NC9594719)

12. Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: SH30256FS)

1. NaCl (2.5 M) (see Recipes)

2. 1 M Tris, pH 7.5-8.0 (see Recipes)

3. 10× annealing buffer (see Recipes)

4. 20× glycerol-tolerant gel buffer (see Recipes)

5. 15% polyacrylamide glycerol-tolerant gel (see Recipes)

Recipes

1. NaCl (2.5 M)

   Reagent	Final concentration	Quantity
   NaCl (powder)	2.5 M	1.46 g
   Nuclease-free H2O	n/a	10 mL
   Total	2.5 M	10 mL




2. 1 M Tris, pH 7.5-8.0

   Reagent	Final concentration	Quantity
   Tris base	1M	121.1 g
   Nuclease-free H2O	n/a	800 mL
   Concentrated HCl	n/a	65 mL
   Nuclease-free H2O	n/a	Add to bring final volume to 1 L
   Total	n/a	1 L




3. 10× annealing buffer

   Reagent	Final concentration	Quantity
   Tris, pH 7.5–8.0 (1 M) (Recipe 2)	100 mM	400 µL
   NaCl (2.5 M) (Recipe 1)	500 mM	800 µL
   EDTA (0.5 M)	10 mM	80 µL
   Nuclease-free H2O	n/a	2,720 µL
   Total	n/a	4 mL

   Store at -20 °C




4. 20× glycerol-tolerant gel buffer

   Reagent	Final concentration	Quantity
   Tris base	1.78 M	216 g
   Taurine	570 mM	72 g
   EDTA (0.5 M)	1 mM	2 mL
   Nuclease-free H2O	n/a	Bring to 1,000 mL
   Total	n/a	1,000 mL

   Store at 4 °C




5. 15% polyacrylamide glycerol-tolerant gel (see Note 4)

   Reagent	Final concentration	Quantity
   Acrylamide/Bis solution 29:1 (30%)	15%	7.5 mL
   Glycerol-tolerant gel buffer (20×) (Recipe 4)	1×	750 µL
   Ammonium persulfate (10%)	0.1%	150 µL
   TEMED	1%	15 µL
   Nuclease-free H2O	n/a	6.6 mL
   Total	n/a	900 mL

Laboratory supplies

1. Standard pipette tips with a volume capacity of 2 µL, 20 µL, 200 µL, and 1 mL

2. Manual pipettes set of 2, 20, 200, and 1,000 µL (Mettler-Toledo, Rainin, catalog number: 17014393, 17014392, 17014391, and 17014382 or similar)

3. Premium microcentrifuge tubes 1.5 mL (Fisherbrand, catalog number: 05-408-129, or similar RNase-free microcentrifuge tubes)

4. Bel-Art^® gel staining box with cover (Millipore Sigma, catalog number: BAF135511000-1EA)

Equipment

1. Standard dry block heaters (VWR, catalog number: 75838-318)

2. Criterion empty cassettes (Bio-Rad, catalog number: 3459901)

3. Criterion cell and PowerPac basic power supply (Bio-Rad, catalog number: 1656019)

4. VWR^® compact UV transilluminator (VWR, catalog number: 76407-432)

5. Laboratory shaker (Reliable Scientific, model: 55)

6. High-resolution digital camera (iPhone, Android, or other cell phone cameras are acceptable)

Software and datasets

1. ImageJ is a Java-based (runs on Mac OS X, Linux, and Windows) freeware available for download at: http://rsb.info.nih.gov/ij/

Procedure