Luckau Protocols:1Kb Ladder: Difference between revisions
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==Purpose== | ==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 | 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== | ||
===Invitrogen 1Kb Ladder=== | ===Invitrogen 1Kb Plus DNA Ladder=== | ||
* catalog# | * catalog# 10787-018 | ||
* | * recommended use: 0.1µg ladder / mm width of well | ||
* STK at 1 | * STK at 1 µg/µL | ||
* [[Media:Invitrogen_1KbLadderProductManual.pdf]] | * [[Media:Invitrogen_1kb_plus.pdf]] | ||
:<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> | |||
===Promega Blue/Orange Loading Dye, 6x=== | ===Promega Blue/Orange Loading Dye, 6x=== | ||
* catalog# G1881 | * catalog# G1881 | ||
* use at 1x concentration | * 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=== | ===Other Knowns=== | ||
* well width ( | * well width (21-tooth comb) = 7mm | ||
:: calculations made for 6mm width, since they end up being more convenient | |||
| Line 37: | Line 51: | ||
===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 | ||
* Per well: 0. | * <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> | ||
* 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=== | ||
120 µL ladder + 880 µL H<sub>2</sub>O + 200 µL loading dye = 1200 µL ladder mix | |||
==Ladder Map== | |||
: [[Image:Invitrogen1Kbv1KbPlusLadder.jpg|300 px]] | |||
==Migration of | ==Migration of Gel Load Dyes (using old ladder)== | ||
* Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp) | * Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp) | ||
: [[Image:AgaroseGel_2_XCBPB | : [[Image:AgaroseGel_2_XCBPB.jpg|100 px]] | ||
Latest revision as of 12:51, 20 December 2012
| 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 transfer to the gel.
Materials
Invitrogen 1Kb Plus DNA Ladder
- catalog# 10787-018
- recommended use: 0.1µg ladder / mm width of well
- STK at 1 µg/µL
- Media:Invitrogen_1kb_plus.pdf
- (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
- [math]\displaystyle{ 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]
- 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)
