Luckau Protocols:1Kb Ladder

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==Purpose==
==Purpose==
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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.
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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==
==Materials==
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===Invitrogen 1Kb Ladder===
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===Invitrogen 1Kb Plus DNA Ladder===
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* catalog# 15615-016
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* catalog# 10787-018
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* Invitrogen says to use: 0.1µg ladder / mm width of well
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* recommended use: 0.1µg ladder / mm width of well
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* STK at 1.0µg/µL
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* STK at 1 µg/µL
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* [[Media:Invitrogen_1KbLadderProductManual.pdf]]
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* [[Media:Invitrogen_1kb_plus.pdf]]
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:<small>(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg]], but they have since discontinued this product. The two products are compatible.)</small>
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===Promega Blue/Orange Loading Dye, 6x===
===Promega Blue/Orange Loading Dye, 6x===
* catalog# G1881
* catalog# G1881
* use at 1x concentration
* use at 1x concentration
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* contains:
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:: 0.4% orange G
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:: 0.03% bromophenol blue
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:: 0.03% xylene cyanol FF
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:: 15% Ficoll® 400 (for density; replaces glycerol)
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:: 10mM Tris-HCl (pH 7.5)
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:: 50mM EDTA (pH 8.0)
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* In a 0.5–1.4% agarose gel in 0.5X TBE:
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:: xylene cyanol FF migrates at approximately 4kb
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:: bromophenol blue at approximately 300bp
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:: orange G at approximately 50bp.
===Other Knowns===
===Other Knowns===
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* well width (24-tooth comb) = 6mm
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* well width (21-tooth comb) = 7mm
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:: calculations made for 6mm width, since they end up being more convenient
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===Calculations===
===Calculations===
* 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
* 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
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* Per well: 0.6µL ladder + 4.4µL H2O + 1µL loading dye = 6µL total ladder mix
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* <math>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>
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* Per well: 0.6 µL ladder + 4.4 µL H<sub>2</sub>O + 1 µL loading dye = 6 µL ladder mix
===Make Ladder Mix===
===Make Ladder Mix===
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120µL ladder + 880µL H2O + 200µL loading dye = 1200µL ladder mix
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120 µL ladder + 880 µL H<sub>2</sub>O + 200 µL loading dye = 1200 µL ladder mix
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==Ladder Map==
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: [[Image:Invitrogen1Kbv1KbPlusLadder.jpg|300 px]]
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==Migration of Gel Load Dyes (using old ladder)==
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* Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)
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: [[Image:AgaroseGel_2_XCBPB.jpg|100 px]]

Current revision

1Kb Ladder Mix

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


Contents

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

(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, Media:Invitrogen_1KbLadderProductManual.pdf, Media:1kb Gel Ladder Map.jpg, 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
  • 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}
  • 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)
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