Luckau Protocols:1Kb Ladder

1Kb Ladder Mix

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Purpose

Nucleic acid ladder is used as a size standard in agarose gel electrophoresis. It contains DNA molecules of known lengths. The ladder is pre-mixed with gel load buffer (color dyes with varying mobility on an agarose gel) for direct transfer to the gel.

Materials

Invitrogen 1Kb Plus DNA Ladder

• catalog# 10787-018
• recommended use: 0.1µg ladder / mm width of well
• STK at 1 µg/µL
• Media:Invitrogen_1kb_plus.pdf

(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, Media:Invitrogen_1KbLadderProductManual.pdf, 300 px, but they have since discontinued this product. The two products are compatible.)

Promega Blue/Orange Loading Dye, 6x

• catalog# G1881
• use at 1x concentration
• contains:

0.4% orange G

0.03% bromophenol blue

0.03% xylene cyanol FF

15% Ficoll® 400 (for density; replaces glycerol)

10mM Tris-HCl (pH 7.5)

50mM EDTA (pH 8.0)

• In a 0.5–1.4% agarose gel in 0.5X TBE:

xylene cyanol FF migrates at approximately 4kb

bromophenol blue at approximately 300bp

orange G at approximately 50bp.

Other Knowns

• well width (21-tooth comb) = 7mm

calculations made for 6mm width, since they end up being more convenient

Protocol

Calculations

• 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
• [math]\displaystyle{ 6 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.6 \mbox{ } \mu \mbox{L ladder per well} }[/math]
• Per well: 0.6 µL ladder + 4.4 µL H₂O + 1 µL loading dye = 6 µL ladder mix

Make Ladder Mix

120 µL ladder + 880 µL H₂O + 200 µL loading dye = 1200 µL ladder mix

Ladder Map

Migration of Gel Load Dyes (using old ladder)

• Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)