(Difference between revisions)
 Revision as of 14:45, 24 October 2012 (view source)← Previous diff Revision as of 18:09, 24 October 2012 (view source)Next diff → Line 26: Line 26: - :::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], but they have since discontinued this product. The two products are compatible.) + ::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg|300 px]], but they have since discontinued this product. The two products are compatible.) Line 46: Line 46: ===Other Knowns=== ===Other Knowns=== * well width (21-tooth comb) = 7mm * well width (21-tooth comb) = 7mm + :: calculations made for 6mm width, since they end up being more convenient Line 51: Line 52: ===Calculations=== ===Calculations=== - * 7mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.7µL ladder per well + * 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well - * $7 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.7 \mbox{ } \mu \mbox{L ladder per well}$ + * $6 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.6 \mbox{ } \mu \mbox{L ladder per well}$ * Per well: 0.6 µL ladder + 4.4 µL H2O + 1 µL loading dye = 6 µL ladder mix * Per well: 0.6 µL ladder + 4.4 µL H2O + 1 µL loading dye = 6 µL ladder mix Line 59: Line 60: - ==Migration of gel load dye & Ladder Map== + ==Ladder Map== + : [[Image:Invitrogen1Kbv1KbPlusLadder.jpg]] + + + ==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.jpg]]    -------------      [[Image:1kb Gel Ladder Map.jpg|300 px]] + : [[Image:AgaroseGel_2_XCBPB.jpg|100 px]]

## 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, 300 px, but they have since discontinued this product. The two products are compatible.)

• 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
• $6 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.6 \mbox{ } \mu \mbox{L ladder per well}$