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 addition of PCR product (or genomic DNA) and transfer to the gel.

Materials

Invitrogen 1Kb Ladder

• catalog# 15615-016
• Invitrogen says to use: 0.1µg ladder / mm width of well
• STK at 1.0µg/µL
• Media:Invitrogen_1KbLadderProductManual.pdf

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 (24-tooth comb) = 6mm

Protocol

Calculations

• 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
• Per well: 0.6µL ladder + 4.4µL H2O + 1µL loading dye = 6µL total ladder mix

Make Ladder Mix

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

Migration of gel load dye & Ladder Map

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

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