INACTIVE DynaMarker, Prestain Marker for Small RNA Plus (75ul)

Product#: xxxDM253S
$165.00
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DynaMarker, Prestain Marker for Small RNA Plus (75 ul) 

Cat. No DM253S 
Size 75 ul
Storage -20°C
Storage buffer 2 mM Tris-HCl (pH 8.0), 8 mM EDTA, 78 % Formamide

Key Word

  • UPF1
  • ATP-dependent RNA helicase
  • nonsense-mediated mRNA decay
  • phosphorylation
  • RIP-seq of transcriptome-binding sites for phospho-UPF1
  • mRNP composition

Description
miRNA is known as a non-coding small RNA, involved in many biological events. The miRNA is derived from precursor miRNA (pre-miRNA, about 100 nt) which is cleaved by the RNase III enzyme Drosha from pri-miRNA. The DynaMarker® Prestain Marker for Small RNA Plus consists of six prestained single-strand (blue and red) nucleic acids (apparent molecular weights are 100, 75, 50, 40, 30 and 20 bases) and it is visible during electrophoresis.

The DynaMarker® Prestain Marker for Small RNA Plus is suitable for monitoring denaturing polyacrylamide gel electrophoresis and blotting onto membranes. The apparent sizes of bands in DynaMarker® Prestain Marker for Small RNA Plus are in excellent agreement with sizes of non-stained RNAs, 100, 75, 50, 40, 30 and 20 bases in length (about 95 % accuracy, see table 1 and figure 2). The DynaMarker® Prestain Marker for Small RNA Plus is supplied in a ready-to-use mixture and doesn’t require heating or addition of a denaturing agent before use.

The DynaMarker Prestain Markers for RNA are visible during electrophoresis.
This advantage helps you to do RNA electrophoresis in proper condition.

DynaMarker Prestain Marker for Small RNA Plus are highly visible indicators with dual colors of blue and red.

DynaMarker Prestain Marker for RNA High is a highly visible indicator with dual colors of blue and purple.

The apparent sizes of the bands in the...
DynaMarker Prestain Marker for Small RNA Plus are 20, 30, 40, 50, 75 and 100 bases in length.

The apparent sizes of the bands in the...
DynaMarker Prestain Marker for RNA High are 200, 500, 1,000, 2,000, 4,000 and 8,000 bases in length.

The DynaMarker Prestain Markers for RNA are ready-to-use mixture. Do not require heat treatment or denaturing agents.

DM253_Fig1.gifFig1.             DM253_Fig2.gifFig2.
Fig1.Electrophoresis profile of DynaMarker® Small RNA II (left)
Fig2.Electrophoresis profile of DynaMarker® Prestain Marker for Small RNA Plus (5 μl) on 10 % acrylamide containing 7.5 M urea gel with 1× TBE buffer as running buffer(right)

 

5.0%

7.5%

10%

12.5%

15%

17.5%

20%

100 base

105.6%

105.6

101.6

98.4

97.2

93.6

92.6

75*

106.2

104.7

103.5

99.5

98.5

94.7

92.4

50

101.4

101.4

101.1

98.7

97.5

95.0

92.2

40

103.1

102.0

103.2

100.8

100.0

97.4

93.9

30

91.0

96.9

98.2

98.9

99.2

99.5

98.8

20

89.8

95.8

98.2

100.3

101.6

101.4

101.4

Table 1. This shows apparent molecular weights compared with the DynaMarker® Small RNA II, and suitable acrylamide concentrations for electrophoresis of the DynaMarker® Prestain Marker for Small RNA Plus.
A : Recomanded A : Possible
(* 75 base RNA is from a newly synthesized RNA. A 75 base RNA is not included in DynaMarker® Small RNA II.)

DM253_Fig3.gifFig3.

Figure3
Electrophoresis profile of DynaMarker® Small RNA II + 75 base RNA* (1) and DynaMarker® Prestain Marker for Small RNA Plus (2) on 10 % and 15 % acrylamide containing 7.5 M urea gel / 1× TBE.
* 75 base RNA is from a newly synthesized RNA. A 75 base RNA is not included in DynaMarker® Small RNA II.

DM253_Fig4.gif

Figure 4
Left: Electrophoresis profile of DynaMaekr® Prestain Marker for Small RNA Plus (1) and RNA sample (2) on 10 % acrylamide containing 7.5 M urea gel / 1× TBE.
Right: Blotting of (1) and (2) onto the nylon membrane.

 

Quality Control
After 24-hrs incubation of the DynaMarker® Prestain Marker for Small RNA Plus at 37 ℃, no visible degradation of the marker is observed in 10 % polyacrylamide – 7.5 M urea gel electrophoresis.

Recommended loading volumes
Comb Load volume
4-10 mm 5-10 μl
>10 mm >10 μl

Note
? For accurate electrophoretic determination of molecular weights, the DynaMarker® Small RNA II (code # DM192) or DynaMarker® Small RNA II Easy Load (code # DM197) should be used.
? A migration of the DynaMarker® Prestain Marker for Small RNA Plus is optimized to use 10 – 15 % acrylamide gel electrophoresis (see table 1).
? This product is not for agarose gel electrophoresis.

Recommended usage

The DynaMarker® Prestain Marker for Small RNA Plus is suitable for monitoring denaturing acrylamide gel electrophoresis and blotting onto membrane. One example is shown below:
?Electrophoresis and blotting of DynaMarker® Prestain Marker for Small RNA Plus
1) Preparation of 10 % polyacrylamide – 7.5 M urea gel

40 % acrylamide : bis solution 5.0 ml
Urea 9.0 g
10 × TBE 2.0 ml
H2O to 20 ml

After urea is dissolved completely, add 20 μl of TEMED and 160 μl of 10 % ammonium persulfate. Mix quickly then pour the gel into the mold of a vertical gel apparatus.

2) Loading and electrophoresis.
Thaw the DynaMarker® Prestain Marker for Small RNA Plus completely before use. Load the denatured RNA sample and 5 μl of DynaMarker® Prestain Marker for Small RNA Plus into a well and run the gel using 1 × TBE electrophoresis buffer at 20 – 40 V / cm.

3) Transfer the DynaMarker® Prestain Marker for Small RNA Plus and RNA from gel to membrane (figure 3).

3-1) Cut a piece of positive charged nylon membrane slightly larger than the gel. Soak the membrane and four sheets of blotting paper of appropriate size in 0.5 × TBE buffer.
3-2) Place two sheets of blotting paper on the anode platform of the transfer cell.
3-3) Place the membrane on top of the blotting paper.
3-4) Transfer the gel from the glass plate to the top of the membrane and press out any air bubbles. (*Make sure that there are no air bubbles between the membrane and the gel.)
3-5) Place another two sheets of blotting paper onto the gel and set the cathode assembly.
3-6) Transfer for 30 – 60 min at 300 mA.
3-7) After ensuring the marker has transferred successfully onto the membrane, remove both paper and gel. Rinse the membrane in 2 × SSC.
3-8) Fix the RNA to the membrane with a UV crosslinker.
3-9) Cut off the marker lane.
3-10) Carry out northern hybridization.

Electrophoresis of DM253 in 64 x speed


References

  • Joseph Sambrook, and David W. Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press.
  • Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith, and Kevin Struhl (1994-) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
  • Wang Y et al. Ectopic expression of miR156 represses nodulation and causes morphological and developmental changes in Lotus japonicus. Mol. Genet. Genomics 2015 Apr;290(2):471-84
  • Kurosaki et al. A post-tranlational regulatory switch on UPF1 controls targeted mRNA degradation. Genes & Dev. 2014. 28:1900-1916
  • Nature Structural & Molecular Biology 21, 901–910 (2014) Rbfox3 controls the biogenesis of a subset of microRNAs Kee K Kim, Yanqin Yang, Jun Zhu, Robert S Adelstein & Sachiyo Kawamoto

 

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