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1 Kb Plus DNA Ladder
TABLE OF CONTENTS
PRODUCT DESCRIPTION
TrackItTM 1 Kb Plus DNA Ladder
Ready-LoadTM 1Kb Plus DNA Ladder
1Kb Plus DNA Ladder
Comparison Between 1 Kb DNA Ladder and 1 Kb Plus DNA Ladder
Quantitation
Source
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
TrackItTM 1 Kb Plus DNA Ladder
Ready-LoadTM 1 Kb Plus DNA Ladder
1 Kb Plus DNA Ladder
T4 DNA Polymerase Labeling Protocol
5' DNA Terminus Labeling Protocol
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
ASSOCIATED PRODUCTS
RELATED TECHNICAL SUPPORT NOTES
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PRODUCT DESCRIPTION
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TrackItTM 1 Kb Plus DNA Ladder
Ready-LoadTM 1Kb Plus DNA Ladder
1Kb Plus DNA Ladder
Comparison Between 1 Kb DNA Ladder and 1 Kb Plus DNA Ladder
Quantitation
Source
TrackItTM 1 Kb Plus DNA Ladder
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The TrackItTM 1 Kb Plus DNA Ladder is supplied in ready-to-load format. The TrackItTM 1Kb Plus DNA Ladder is
suitable for sizing linear double-stranded DNA fragments from 100 bp to 12 Kb.
The TrackItTM DNA Ladders are formulated with unique tracking dyes, Xylene Cyanol FF (XCFF) and Tartrazine
that allow you to visually track DNA migration during electrophoresis and also indicate when maximum resolution
is achieved. The tracking dyes do not obscure the visualization of DNA bands in the ladders as the dyes run outside
the limits of most DNA bands in the ladder.
The TrackItTM 1 Kb Plus is supplied at 10 mM Tris-HCl, pH 7.5; 10 mM EDTA, pH 8.0; 0.06% XCFF; 0.6%
tartrazine; 5% glycerol; 5 mM NaCl
Ready-LoadTM 1 Kb Plus DNA Ladder
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The Ready-LoadTM 1 Kb Plus DNA Ladder is a ready-to-load version of the 1Kb Plus DNA Ladder. Like the
other Ready-LoadTM ladders, the product is supplied in loading buffer and is ready to load directly onto agarose
gels. This product is similar to the 1Kb Plus DNA Ladder in that it contains twelve bands ranging in size from
1,000 bp to 12,000 bp in exact 1,000 bp increments. In addition to these 12 bands, the ladder contains eight
bands ranging in size from 100 鈥? 1,650 bp.
Ready-LoadTM 1 Kb Plus DNA Ladder is supplied at 0.1 ug/uL in 10 mM Tris Hcl (pH 7.5), 10 mM EDTA,
0.05 % bromophenol blue, 5% glycerol, 5mM NaCl.
1 Kb Plus DNA Ladder
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The 1 Kb Plus DNA Ladder is suitable for sizing linear double-stranded DNA fragments from 100 bp to 12
Kb. The bands of the ladder each contain from 1 to 12 repeats of a 1000-bp DNA fragment. In addition to these
12 bands, the ladder contains eight bands that range from 100 to 1,650 bp.
The 1 Kb DNA Ladder is supplied at 1 ug/ul in 10 mM Tris HCl (7.5), 50 mM NaCl, 1.0 mM EDTA.
The 1,650-bp band contains approximately 8% of the mass applied to the gel.
Comparison Between 1 Kb DNA Ladder and 1 Kb Plus DNA Ladder
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1 Kb Plus DNA Ladder 1 Kb DNA Ladder
Low molecular weight bands in 50 and Low molecular weight bands in variable size
100 bp increments (see below) increments
The 1 Kb repeats are 1 Kb The 1Kb repeats are 1018 bp repeats
Low molecular weight bands are mostly LMW bands are less intense and not as useful
same intensity as 1 Kb bands
There are bands between the 500bp and There is a gap between the 506 and
1000bp bands (650 and 850bp) 1018 bp band
All bands have the same overhang (GATC) Some bands are blunt and some bands have
an Ava I overhang
Orientation band at 1650 bp Orientation band at 1636 bp
20 bands 18 bands are easily detected
1 Kb Plus DNA Sizes (bp):
100, 200, 300, 400, 500, 650, 850, 1000, 1650, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000.
Quantitation
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The 1Kb Plus DNA ladders are not designed to quantitate DNA samples. The total concentration of the ladder is
measured via absorbance (before mixing with the Ready-to-Load buffer). However, there is not an accurate estimation
of the mass of each band.
Source
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The 1650 bp band is generated from pUC. The bands smaller than 1000 bp are derived from lambda DNA.
SHIPPING CONDITIONS
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Ladders may be shipped at RT or on wet ice.
STORAGE CONDITIONS
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1 Kb Plus DNA Ladder should be stored at -20oC.
Ready-LoadTM and TrackItTM Ladders should be stored at either 4oC or R.T.
STABILITY
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1 Kb Plus DNA Ladder is stable for >1 year when stored at -20oC.
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Ready-LoadTM and TrackItTM Ladders are stable at least 6 months at all temps.
QC SPECIFICATIONS
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Agarose gel analysis shows that all bands are distinguishable and show essentially equal intensity by ethidium
bromide staining. The A260/A280 ratio is in the range of 1.8-2.0.
PROTOCOL AND APPLICATION NOTES
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TrackItTM 1 Kb Plus DNA Ladder
Ready-LoadTM 1 Kb Plus DNA Ladder
1 Kb Plus DNA Ladder
T4 DNA Polymerase Labeling Protocol
5' DNA Terminus Labeling Protocol
TrackItTM1 Kb Plus DNA Ladder
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DO NOT HEAT BEFORE LOADING.
For TAE or TBE 0.8-1% agarose gels load 5 ul TrackItTM DNA Plus Ladder per 5mm lane width.
For 0.8% agarose E-gel, mix 2ul TrackItTM DNA Plus Ladder with 18ul water and load 20 ul of the diluted
ladder per well of a single-comb E-gel.
After electrophoresis, stain the DNA ladder with ethidium bromide or SYBR庐 Green I Nucleic Acid Gel Stain.
Note: There is no need to stain E-Gel庐 agarose gels as the gels contain ethidium bromide.
Ready-LoadTM 1Kb Plus DNA Ladder
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Recommended load is 1 ul of ladder per mm of lane width (typically 5 ul). The ladder can be diluted in 10X
BlueJuice Gel Loading Buffer and used at a final concentration of 2X for electrophoresis of DNA standards
(catalog # 10816015, 10X BlueJuice).
After electrophoresis, stain the DNA ladder with ethidium bromide or SYBR庐 Green I Nucleic Acid Gel Stain.
During 1% agarose gel electrophoresis with Tris-acetate (pH 7.5) as the running buffer, bromophenol blue
migrates together with the 500 bp band.
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P should not be used to label the ladder because the product contains 5% glycerol which inhibits enzymatic
activity.
If using a probe that is pUC based or has some pUC or Lambda DNA sequence as a probe for a Southern blot,
you may observe hybridization to one or some of the 1 Kb Plus ladder bands. The 1650 band is from pUC.
Other bands are derived from sequences of Lambda DNA.
1 Kb Plus DNA Ladder
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T4 DNA Polymerase Labeling Protocol
5' DNA Terminus Labeling Protocol
Apply approximately 0.1 ug of the ladder per mm lane width. The ladder can be diluted in10X BlueJuice Gel
Loading Buffer and used at a final concentration of 2X for electrophoresis of DNA standards (catalog #
10816015, 10X BlueJuice).
DO NOT HEAT BEFORE LOADING.
Alternatively, dilute in TE containing a final concentration of 20 mM NaCl if running on agarose gels. Apply
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approximately 0.1 碌g of ladder per mm lane width.
After electrophoresis, stain the DNA ladder with ethidium bromide or SYBR庐 Green I Nucleic Acid Gel Stain.
The 1 Kb PLUS DNA ladder can be radioactively labeled by one of the following methods.
T4 DNA Polymerase Labeling Protocol (see protocol below).
5' DNA Terminus Labeling Protocol (see protocol below).
T4 DNA Polymerase Labeling Protocol for 1 Kb Plus DNA Ladder
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1. Exonuclease Reaction (Degradation of DNA from both 3' ends)
To a 1.5-ml microcentrifuge tube on ice, add the following:
5X T4 DNA polymerase reaction buffer* 4 ul
1 Kb Plus DNA Ladder 10 ug
T4 DNA polymerase 40 units
Autoclaved water to 20 ul
*[165 mM Tris acetate (pH 7.9), 330 mM sodium acetate, 50 mM magnesium acetate,
2.5 mM DTT, 500 ug/ml BSA]
Make sure all components are at the bottom of the tube. Mix thoroughly but not vigorously. Centrifuge
briefly.
Incubate 2 min at 37oC. (about 25 nucleotides/min are removed). Cool reaction vial on ice.
2. Resynthesis Reaction (Fill-in) This reaction will resynthesize the degraded DNA strands.
Place into the reaction vial which is sitting in ice after the exonuclease reaction:
Autoclaved water 8 ul
5X T4 DNA polymerase reaction buffer 6 ul
dCTP (2 mM 5 ul
dGTP (2 mM 5 ul
dTTP (2 mM 5 ul
[alpha-32P]dATP (3000 Ci/mmol; 10 mCi/ml 1 ul
Mix thoroughly. Centrifuge briefly. Incubate 2 min at 37oC, then add 5 ul of 2 mM dATP.
Incubate 2 min at 37oC. Stop reaction by adding 2.5 ul of 0.5 M EDTA. Centrifuge for 10 s.
The cpm incorporated is determined by adding 1 ul of reaction to 24 ul of 250 mM NaCl, 25 mM EDTA. Spot
5 ul of dilution on a glass fiber filter. Place filter in 10% (w/v) TCA + 1% (w/v) pyrophosphate. Wash filter 3
times with 5%(w/v) TCA and then 2 times with ethanol. The filter is dried and then counted using an
appropriate scintillant.
Add 5 ul 0.1% (w/v) bromophenol blue, 0.1 mM EDTA, 50% (v/v) glycerol to the sample. Load 1 x 105 cpm
in a lane.
5' DNA Terminus Labeling Protocol for 1 Kb Plus DNA Ladder (Phosphate Exchange Reaction)
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This reaction will yield specific activities of approximately 1-5 x 105 cpm/pmol of ends.
Add the following components to a 0.5-ml microcentrifuge tube in the following order:
Autoclaved water 11 ul
1 Kb Plus DNA Ladder (5 ug 5 ul
5 X exchange reaction buffer [250 mM Imidazole-HCl pH 6.4,
60 mM MgCl2, 5 mM BMeOH, 350 uM ADP] 5 ul
[gamma-32P]ATP (10 碌Ci/ul) 3 ul
T4 polynucleotide kinase (5 or 10 U/ul) 1 ul
Incubate the reaction mixture at 37oC for 30 minutes. Increasing reaction times beyond 30 min will not
increase labeling of the DNA.
Stop reaction by adding 1 ul of 0.5 M EDTA. Centrifuge for 10 s.
Determine radioactive incorporation. The cpm incorporated is determined by adding 1 ul of reaction to 24 ul
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of 250 mM NaCl, 25 mM EDTA. Spot 5 ul of dilution on a glass fiber filter. Place filter in 10% (w/v) TCA +
1% (w/v) pyrophosphate. Wash filter 3 times with 5%(w/v) TCA and then 2 times with ethanol. The filter is
dried and then counted using an appropriate scintillant.
Add 5 ul 0.1% (w/v) bromophenol blue, 0.1 mM EDTA, 50% (w/v) glycerol to the sample.
Load 1 x 105 cpm in a lane.
ALTERNATE PRODUCTS AND COMPATIBILITY
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Name Size Part Number Catalog Number
TrackItTM 1 Kb DNA Ladder 100 Apps 95615 10488-072 (10488072)
Ready-LoadTM 1 Kb DNA 100 Apps 90487 10381-010 (10381010)
Ladder
1 Kb DNA Ladder 250 ug 95615 1561-5016 (15615016)
1 Kb DNA Ladder 1 mg 95615 15615-024 (15615024)
PRODUCT DOCUMENTATION
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Invitrogen Catalog
Brochures Citations Cell lines
COA FAQ Licensing
Manuals MSDS Newsletters
Vector Data
PRODUCT NAME & CATALOG NUMBER
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Name Size Part Number Catalog Number
TrackItTM 1 Kb Plus DNA 100 apps 55382 10488-085 (10488-085)
Ladder
Ready-LoadTM 1 Kb Plus 100 apps 54385 12308-011 (12308-011)
DNA Ladder
1 Kb Plus DNA Ladder 250 ug 50842 10787-018 (10787018)
1 mg 10787-026 (10787026)
ASSOCIATED PRODUCTS
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BlueJuiceTM Gel Loading Buffer, Catalog No. 10816-015
TrackItTM Cyan/Yellow Loading Buffer, Catalog No. 10482-035
SYBR庐 Green I Nucleic Acid Gel Stain, Catalog No. S7563
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