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DIG Easy Hyb Standard buffer + 50% formamide

DIG-Labeled Probe Synthesis

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Digoxigenin labelled probe synthesis - Carroll lab

Originally nitrocellulose membranes were used but their fragility prompted to search for alternative types of support matrix, resulting in the introduction of nylon membranes in the early 1980s. The main advantages of nylon membranes is their greater tensile strength and that DNA can be bound covalently by UV cross-linking. Nylon membranes can therefore be reprobed up to about 12 times without becoming broken or losing their bound DNA. In contrast nitrocellulose membranes are fragile (allow only 3 times reprobing) and do not bind DNA covalently: baking to immobilize the DNA leas to a relatively weak hydrophobic attachment through exclusion of water. The disadvantage of nylon membranes is the amount of background signal seen after hybridization. On the other site nylon membranes are able to bind about five times more DNA per cm2 than nitrocellulose. Nylon retains DNA fragments down to 50 nucleotides in length, but nitrocellulose is inefficient with molecules

To generate DIG labeled probe (35 cycles, 94C 30 seconds, 55C 30 seconds and 72C, 1 minute/1 kb):

unit = 20 ug/ml)
DNA fragment chopped into pieces of
Klenow fragment of DNA polymerase I
tRNA at 25 mgs/ml
EDTA 0.5 M pH 8
PBT (PBS + 0.1% Tween 20)(PBS = 10 mM KPO4; 140 mM NaCl pH 7.2)
Centricon 10 (Amicon) 1) Combine: 100 ng (embryo probes) or 400 ng (disc probes) fragmented DNA
2 ul pDN6 (at 25 mgs/ml)
deionized H2O to 9 ul

2) Boil for 2 min.; quick chill in ice water 3) Add : 2 ul 10x hexanucleotide mix
2 ul 10x nucleotides
6 ul H2O
1 ul Klenow 4) Place in 16oC water bath overnight.

MOENS LAB -- Totally Anal RNA in situ Probe Synthesis

Several alternatives are available for the detection of digoxigenin (or biotin)-labeled probes.

You may use sealable containers or heat sealable plastic bags for the hybridization if using DIG Easy Hyb solutions. Roller tubes in combination with a hybridization oven may also be used for standard buffers.

Use only one primer. We set up two reactions, one to generate a sense probe with 5'primer and the other to generate an anti-sense probe with 3'primer. RNA hybridization with the sense probe will be a background control.

Dig RNA Probe Synthesis and Purification - …

DIG Easy Hyb Standard buffer Standard buffer + 50% formamide High SDS buffer

Dilute boiled probe in HB to give 100 l of solution. Try a 1:2 dilution for probes to messages of unknown abundance. Experience with probes suggests that probe dilution is not critical – a strong, clean signal was obtained at dilutions between 1:2 and 1:5, but it began to fall off at 1:10. Probe to major sperm protein RNA, which is very abundant during spermatogenesis, could be diluted 1:20 without any fall off in signal.

In the following chapters we give recommendations for probe concentrations in the different applications. These recommendations refer to newly synthesized, DIG-labeled probe. The labeling efficiency has always to be confirmed by estimating the yield of a labeling reaction. The recommended probe concentration must be regarded as a starting point for your hybridization.

DIG Easy Hyb*
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  • RNA probe synthesis - University of Manitoba

    For further information on products to label and detect digoxigenin-modified nucleic acids refer to

  • -labeled RNA probe in situ hybridization protocol

    The digoxigenin-anti-digoxigenin system uses digoxigenin (DIG), a cardenolide steroid isolated from Digitalis plants.

  • Digoxigenin Oligo Labeling - Bio-Synthesis, Inc.

    RNA Probe Synthesis

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DIG pcr labeling and northern blot - ..

We have found that DIG-labeled probes demonstrate the same hybridization kinetics as radiolabeled probes. Hybridization and washing conditions for DIG-labeled probes do not differ substantially from those of radiolabeled probes.

Making RNA probes with T7 transcription - …

Figure 7: Mock hybridization and effect probe concentration. Naked pieces of membrane were incubated with the indicated amounts of DIG-labeled DNA probe and detected with chemilumininescence.

Making RNA probes with T7 transcription

One of the advantages of the DIG-System is the stability of the labeled probe. After hybridization against the blotted target, the hybridization solution still contains large amounts of unannealed DIG-labeled probe. Simply pour the solution into a plastic tube and store at –20° C for DNA probes and –70°C for RNA probes. DIG labeled probes are stable for at least 1 year when stored in this manner. For reuse, thaw and denature by heating to +95°C for 10 min. If the hybridization solution contains formamide or .

In vitro T7 transcription of an RNA probe

The DIG System is an effective system for the labeling and detection of DNA, RNA, and oligonucleotides. The protocols for labeling with digoxigenin (Figure 1) and subsequent detection are based on well established, widely used methods. DNA, RNA, and oligonucleotide probes are labeled according to the methods (usually enzymatic) used for preparing radioactive probes. Hybridization of digoxigenin-labeled probes (e.g., to target DNA or RNA on a Southern or Northern blot) is also carried out according to standard protocols, except that a special blocking reagent is used to eliminate background. The signal on the nucleic acid blot is detected according to the methods developed for western blots. The incorporation and spacing of digoxigenin in DNA, RNA, and oligonucleotides can be varied by using different labeling protocols.

(Figure 1 A) was prepared using a PCR DIG probe synthesis ..

Split-signal fluorescence insitu hybridization analysis of ETV6 rearrangement in patientswith acute promyelocytic leukemia. Red and green fluorescencesignals correspond to the labeled probes DIG525I23 and Bio407P10,respectively. (A) Red and green fluorescence signal co-localizationsuggested no ETV6 rearrangement. (B) Separation of one red and onegreen fluorescence signal suggested ETV6 rearrangement. (C)Separation of both red and green signals suggested ETV6rearrangement on homologous chromosomes. (D) Metaphase chromosomeswere clearly recognizable. Both chromosomes 12p and 1q exhibitedfluorescence signals, suggesting that there was translocation onhomologous chromosomes between 12p and 1q (magnification, ×1,000).ETV6, Ets variant 6.

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