Luckau Protocols:1Kb Ladder

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(New page: Ladder Mix [edit]Manufacturer specifications 0.1µg ladder / mm width of well STK at 1.0µg/µL Media:Invitrogen_1KbLadderProductManual.pdf [edit]Other Knowns well width (24-tooth comb) =...)
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Ladder Mix
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| width="600" style="background-color: #BCED91;" align="center"| <span style="font-size:32px;"> 1Kb Ladder Mix </span>
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[edit]Manufacturer specifications
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0.1µg ladder / mm width of well
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<span style="font-size:16px;"> [[User:Tara_K._Luckau | Tara K. Luckau's Home Page]] </span>
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STK at 1.0µg/µL
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Media:Invitrogen_1KbLadderProductManual.pdf
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[[User:Tara_K._Luckau/Notebook/Team_ConGen | Conservation Genetics Lab Notebook]]
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[edit]Other Knowns
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well width (24-tooth comb) = 6mm
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[[Luckau_Protocols | Tara's Protocols]]
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Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye
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[edit]Calculations
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6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
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==Purpose==
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Per well: 0.6µL ladder + 4.4µL H2O + 1.5µL loading dye = 6.5µL total ladder mix
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(loading dye = xylene cyanol and bromophenol, made by Bohonak lab)
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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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[edit]Make Ladder Mix
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120µL ladder + 880µL H2O + 300µL loading dye = 1300µL ladder mix
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==Materials==
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===Invitrogen 1Kb Ladder===
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* catalog# 15615-016
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* Invitrogen says to use: 0.1µg ladder / mm width of well
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* STK at 1.0µg/µL
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* [[Media:Invitrogen_1KbLadderProductManual.pdf]]
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===Promega Blue/Orange Loading Dye, 6x===
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* catalog# G1881
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* use at 1x concentration
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===Other Knowns===
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* well width (24-tooth comb) = 6mm
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==Protocol==
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===Calculations===
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* 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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===Make Ladder Mix===
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120µL ladder + 880µL H2O + 200µL loading dye = 1200µL ladder mix

Revision as of 23:50, 2 May 2011

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


Materials

Invitrogen 1Kb Ladder

Promega Blue/Orange Loading Dye, 6x

  • catalog# G1881
  • use at 1x concentration

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

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