1. Lab Safety
Gloves (optional) and safety glasses (yes)
Labcoats – optional, buy your own
Using pipettemen (small volume pipetting)
Other pipettes (larger volumes)
Gels: agarose vs polyacrylamide; resolution
running buffers and loading buffers
visualizing DNA
Lab exercise; pouring gel, mixing samples and running gel

2. What is BSL1?

3. Starch gel electrophoresis
Separation of proteins by size
Resolution not so good
Smithies, O., Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults (1955) Biochem J 61: 629
Oliver Smithies

4. agarose vs polyacrylamide
For separation of DNA/RNA and protein molecules by size
Resolution – agarose gels, usually 0.7 – 2.0 % horizontal gels
50 bp to kb
Higher the concentration – better resolution of smaller sizes
Lower the concentration – better resolution of larger sizes
Higher concentration – longer runtime
Voltage V common, heat can be an issue (melted gels/equipment)
RNA gels also made with agarose
PAGE (polyacrylamide gel electrophoresis) vertical gels
Polyacrylamide gels – base pair resolution
Sequencing gel 6% gel 19:1 acrylamide:bis-acrylamide + 6 M urea
Protein PAGE gels also, denaturing/non-denaturing

5. Setting up an agarose gel
Put combs in gel mold, dams if needed
Measure out buffer (120 ml) into flask
Add agarose (1.2 g, ~1 % gel)
Boil (hot plate or microwave)
Cool to touch
Pour and wait to solidify
Remove combs, gently
Add buffer sufficient to cover gel
Load samples, run
Stain ~ 5 min with EtBr ( 2 mg/ml)
Visualize with UV light source in dark

6. Prepare a 5X stock solution in 1 L of H2O: 54 g of Tris base
TBE buffer
Prepare a 5X stock solution in 1 L of H2O:
54 g of Tris base
27.5 g of boric acid
20 mL of 0.5 M EDTA (pH 8.0)
The 0.5X working solution is 45 mM Tris-borate/1 mM EDTA.
$5.39 for 76 oz
Borax gel prep
10 X stock of borax (100 mM)
19 g of borax in 500 ml, resulting pH 9.3
Assuming Na2B4O7·10H2O, MW=
Use 1X (10 mM) for agarose gel
and running buffer (pH 9.0)
Based on BioTechniques 36:214
76 oz = 2.1 kg
Enough for 56 liters of 10X stock
= 560 liters of buffer
~ ml 0.05c/gel
50X TAE (1 $50) from
Boston Bioproducts
= 50 liters of buffer
~ ml 5c/gel

7. DNA Markers Ladders (1 kb, 100 bp, etc)
1 kb ladder (Boston Bioproducts)
Buffered/EDTA solution of high range DNA
ladder (10, 8, 6, 5, 4, 3, 2, and 1, 0 kb)
Restriction digests
of genomes
(plasmids or phage primarily)
1 kb DNA ladder

8. 6X gel loading buffer
Bromophenol blue 0.25%, Xylene cyanol 0.25%, and Glycerol 40% in autoclaved DI water
Can also use sucrose for density
Can leave out BPB or XC
Xylene cyanol – size marker
10,000 bp at 0.7% agarose
4000 bp at 1.5%
750 bp at 2.0%
200 bp at 3.0%
Bromophenol blue – size marker
1000 bp at 0.7% agarose
500bp at 1%
150bp at 2%
50bp at 3%

9. Many options EtBr, SYBR Green, GelRed,…
Staining the gel
Many options EtBr, SYBR Green, GelRed,…
Ethidium bromide (EtBr) intercalating dye
10 mg/ml (Sigma, BB, others; cheap)
5 ml > 25 ml running buffer = 2 mg/ml working solution

10. 1 kb ladder ml
Lambda genome DNA, uncut ml
N. crassa genome DNA, uncut ml
pUC ml
pBSSK ml
pGREEN ml
pCRISPR ml