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Invitrogen™ Millennium™ RNA Markers-Formamide

Catalog No. AM7151
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AM7151 50 μL
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Catalog No. AM7151 Supplier Invitrogen™ Supplier No. AM7151
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Description

For accurate size determination of single-stranded RNA transcripts from 0.5 to 9kb and can be used in any Northern protocol

Sufficient markers are provided for 25 Northern gel lanes. They are a mixture of 10 discrete RNA transcripts with easy-to-remember sizes: 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 9 kilobases. The markers are supplied in formamide and show increased stability and resistance to RNase compared to other large molecular weight RNA markers. They may be stained with ethidium bromide either during or after electrophoresis. Because of their quantity and even spacing, these markers are ideal for constructing very accurate standard curves for mRNA size determination. They also provide a reference scale for easy verification and comparison of known bands. 1-2μg (1-2μL) of RNA Millennium™ Markers will generate 10 distinct bands on a 1% denaturing agarose gel with ethidium bromide staining. All marker lots undergo stringent nuclease testing and are shown to be stable overnight at 37°C. Compatible with formaldehyde-containing and glyoxal-containing gels.

Agarose Gel Electrophoresis, DNA and RNA Purification and Analysis, Northern Blotting, Nucleic Acid Gel Electrophoresis and Blotting

Order Info

Shipping Condition: Dry Ice

Specifications

Content And Storage Store at below –70°C.
Concentration 1 μg/μl
Ready to Load No
Size Range 0.5 to 9 kb
Gel Compatibility Formaldehyde-Containing Gels, Glyoxal-Containing Gels
Quantity 50 μL
Shipping Condition Dry Ice
Product Line Ambion, Millennium Markers
Product Type RNA Marker-Formamide

Frequently Asked Questions (FAQs)

Why are my RNA bands not sharp?

(1) RNA was not completely denatured. Electrophorese RNA under denaturing conditions. Use urea, formamide or formaldehyde gels, or glyoxal-treated RNA.

(2) For glyoxal-treated RNA, a buffer gradient formed during electrophoresis. Recirculate buffer during electrophoresis to prevent gradient formation.
What is the cause of extra bands when using an RNA ladder?

Extra bands appear in RNA Ladders for a few reasons:

(1) RNA was not completely denatured. Electrophorese RNA under denaturing conditions. Use urea, formamide or formaldehyde gels, or glyoxal-treated RNA.

(2) Extra bands may be a result of using formaldehyde that is not fresh; the pH becomes acidic in older formaldehyde. In our hands, when fresh formaldehyde with neutral pH was used, the extra bands disappeared.

(3) Alternatively, if the extra bands appear after hybridization, it could be that the gel purified probe contains contaminating vector DNA (pUC or pBR) that hybridizes to RNA Ladder template DNA.
Why are some of the RNA marker bands not visible?

Missing RNA bands may be due to:

(1) A small amount of RNA diffused out of gel during extended destaining. Minimize destaining time. Destaining for 2 hours is sufficient for most applications.

(2) RNA bands of similar molecular size were not resolved. Use the correct gel type and denaturing conditions.
Why are the RNA bands disappearing when looking and photographing a gel on a UV box?

RNA was exposed to UV light for extended periods of time. Minimize exposure to UV light. Stain and destain gels in the dark and photograph the gel immediately.
Why are the RNA marker bands so faint?

Many factors could affect the intensity of the bands as summarized below.

(1) Insufficient RNA was loaded on the gel. Increase the amount of RNA loaded.

(2) RNA was degraded. Avoid nuclease contamination of the RNA standards. Store RNA at -70 degrees C in formamide. Deionize formamide and glyoxal, store aliquots at -20 degrees C.

(3) RNA was electrophoresed off the gel. Electrophorese the gel for less time, at a lower voltage, or in a higher percentage gel.

(4) For ethidium bromide-stained RNA, improper UV light source was used. Use short-wavelength (254 nm) UV light. For ethidium bromide-stained RNA, improper staining and destaining conditions were used. Stain in the dark with 5 mg/mL ethidium bromide. For thin (3.2 mm) formaldehyde gels, stain 5 min and destain 1 h. For thicker gels, stain 30 min and destain 2 hr. To reduce background staining, use 0.66 M formaldehyde instead of 2.2 M formaldehyde in gels. For glyoxal RNA gels, stain and destain in 0.5 M ammonium acetate to help reduce background staining.

(5) For radiolabeled RNA, an improper labeling method was used.
What kind of RNA molecular weight ladders do you offer?

We offer the following RNA Ladders:


RiboRuler High Range RNA Ladder (Cat. No. SM1821)
RiboRuler Low Range RNA Ladder (Cat. No. SM1831)
Century Marker Templates, 100-500 bp (Cat. No. AM7780)
Century-Plus Marker Templates, 0.1-1 kb (Cat. No. AM7782)
Decade Markers, 10-150 bp (Cat. No. AM7778)
Millennium Markers-Formamide, 0.5-9 kb (Cat. No. AM7151)
RNA 6000 Ladder, 0.2-6 kb (Cat. No. AM7152)
RNA Century-Plus Markers, 0.1-1 kb (Cat. No. AM7145)
RNA Millennium Markers, 0.5-9 kb (Cat. No. AM7150)
Is formamide (Cat. No. 15515-026) supplied as a liquid? When is deionization required, and can you provide a protocol?

Yes, formamide is supplied as a liquid even though it is sold by weight.

It is appropriate to use out of the bottle for most applications (hybridization buffer, loading buffers for sequencing gels, and formaldehyde-containing RNA gels) if less than one year old and stored properly (-20 degrees C). (If formamide smells like ammonia, it is probably breaking down and should not be used.) However, we have seen that in a protocol we have for running RNA gels without formaldehyde in the gel, freshly deionized formamide is essential for preparation of the loading buffer. That protocol as well as the protocol for deionizing the formamide follows:

These reagents are used in electrophoresis of RNA in agarose gels in 1X MOPS EDTA buffer. (Note: no formaldehyde is used in the gel.) The sample is prepared in the sample buffer (1.5 to 3 µL sample mixed with sample buffer). Heat the RNA in sample buffer for 10 min at 65 degrees C. Place on ice immediately then load gel.

10X MOPS EDTA (10X ME)

(1) Measure out 700 mL DEPC-treated water in a DEPC-treated 1-liter graduated cylinder and place in a DEPC-treated 1-liter beaker.
(2) Place beaker on stir plate and begin stirring with a DEPC-treated stir bar.
(3) Add 104.65 g MOPS to beaker. Weigh out MOPS using a sterile-flamed spatula from an unopened bottle MOPS or one set aside for RNA only. Dissolve by stirring.
(4) Add 40 mL 0.25 M EDTA to solution. Note: the EDTA solution should be made with DEPC-treated processed water.
(5) Adjust pH to 7.0 by adding 10 N NaOH (~20 mL)
(6) Bring up to 1 liter volume with DEPC-treated water.
(7) Filter solution using a 0.2 µm Nalgene filter unit.
(8) Store at 4 degrees C for six months in a DEPC-treated glass bottle. (This prevents solution from yellowing.) NOTE: The presence of yellow color does not affect the performance of the buffer.

Formamide Deionization protocol:
1. Add 1 g mixed bed, ion exchange resin for every 10 ml of formamide. Suitable ion exchange resins include BioRad AG501-X8 and Fisher Resin 1-300.
2. Stir for 30 to 60 min @ RT.
3. Filter through Whatman No. 1 filter paper.
4. Dispense into units of use and store at -20°C
What staining procedures are recommended for RNA ladders?

RNA can, in fact, be stained with ethidium bromide. However, proper staining procedures are important in order to visualize RNA. Insufficient staining and destaining will result in RNA bands being lost in the background. Ethidium bromide can be added to samples prior to electrophoresis.

We recommend the following amounts for this approach, using denaturing gels with formaldehyde contents of 2.2 M:
--For a gel thickness of 6.4 mm, the ethidium bromide should be added to the sample to a final concentration of 1.2 mg/mL.
--For a 3.2 mm thick gel, the final concentration should be 0.13 mg/mL.

If ethidium bromide staining is carried out after electrophoresis, the following recommendations should be followed (ethidium bromide at 5 mg/mL):
--3.2 mm thick gels containing 2.2 M formaldehyde, 5 min staining, 0.5 to 1 hr destaining
--6.4 mm thick gels containing 2.2 M formaldehyde, 5-30 min staining, 2 to 3 hr destaining
--6.4 mm thick gels containing 0.6 M formaldehyde, 10-20 min staining, 1 to 2 hr destaining

For Research Use Only. Not for use in diagnostic procedures.