Luckau Protocols:1Kb Ladder: Difference between revisions
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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 | {|{{table}} width="900" | ||
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| width="600" style="background-color: #BCED91;" align="center"| <span style="font-size:32px;"> 1Kb Ladder Mix </span> | |||
[ | |align="left" style="background-color: #9DB68C;" | | ||
0.1µg ladder / mm width of well | <span style="font-size:16px;"> [[User:Tara_K._Luckau | Tara K. Luckau's Home Page]] </span> | ||
STK at 1.0µg/µL | |||
Media:Invitrogen_1KbLadderProductManual.pdf | [[User:Tara_K._Luckau/Notebook/Team_ConGen | Conservation Genetics Lab Notebook]] | ||
well width (24-tooth comb) = 6mm | [[Luckau_Protocols | Tara's Protocols]] | ||
|} | |||
6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well | ==Purpose== | ||
Per well: 0.6µL ladder + 4.4µL H2O + | |||
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. | |||
120µL ladder + 880µL H2O + | |||
==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 | |||
===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 |
Revision as of 20:50, 2 May 2011
1Kb Ladder Mix |
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
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