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) =...)
Current revision (14:51, 20 December 2012) (view source)
 
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Ladder Mix
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{|{{table}} width="900"
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|-
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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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|align="left" style="background-color: #9DB68C;" |
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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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|}
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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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Per well: 0.6µL ladder + 4.4µL H2O + 1.5µL loading dye = 6.5µL total ladder mix
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==Purpose==
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(loading dye = xylene cyanol and bromophenol, made by Bohonak lab)
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[edit]Make Ladder Mix
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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.
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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 Plus DNA Ladder===
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* catalog# 10787-018
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* recommended use: 0.1µg ladder / mm width of well
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* STK at 1 µg/µL
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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===
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* catalog# G1881
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* 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.
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===Other Knowns===
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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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==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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* <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
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===Make 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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