Information

Can I use TBE to elute a plasmid shipped on filter paper?

Can I use TBE to elute a plasmid shipped on filter paper?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I'm receiving a plasmid shipped to me dried on sterile filter paper, as described in this question. This protocol and many others like it call for eluting the DNA from the paper using TE buffer.

My question is can I also use TBE to elute, as I already have a bunch on-hand? And in general, what concentration should the buffer be as I never see that mentioned in any of the protocols (including the AddGene version).


In my experience it does not matter if you use water, TBE or TE buffer. Even with a little EDTA in it, it will not mess up enzymatic reactions as the concentration is way too low. TBE/TE buffer usually contains 1mM of EDTA which can chelate 1mM of bivalent cations. Since you not use the DNA undiluted (which also binds a lot of magnesium needed for most enzymes btw.) this really doesn't affect you in reality (been there, done that multiple times).

Additionally, I would recommend not using this DNA directly but transform it into bacteria first and then do a proper miniprep so you have enough material to work with (maybe even do a few restriction cuts and run it on a gel, to ensure the right identity) and a stock you can go back to if something goes wrong.


This is not exactly an answer to your question, i.e., how borate ions affect storage of your plasmid DNA.

But I would recommend you to use neither TBE nor TE, as they contain EDTA that might interfere with downstream enzymatic reactions. Use water (ddH2O) instead. As long as you store your plasmid DNA at -20°C it will be perfectly stable for many years. Alternatively, use a simple Tris buffer.

Addgene has a good article on this.


Biomiga Express Plasmid Miniprep Kit (10 mins - 50 preps)

Done shopping? You can create a PDF of your cart for later or for your purchasing dept! Details at checkout.

This helpful video explains the entire process of the PD1218-01 plasmid prep kit: https://vimeo.com/231585099

This express plasmid mini prep kit includes:

Buffer F1, Buffer F2, Buffer F3, DNA Wash Buffer, Buffer KB, Elution Buffer, Lysate Clearance Column, Mini Column and RNase A

Instructions:

1. Harvest 1-2 mL of fresh bacterial culture by centrifugation for 1 minute at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely. 2. 2. Add 200 µL Buffer F1 and completely resuspend bacterial pellet by vortexing or pipetting (Complete resuspension is critical for optimal yields).

3. Add 200 µL Buffer F2, mix by inverting 10 times (do not vortex) and incubate at room temperature for 2 minutes until the solution becomes clear.

4. Add 200 µL Buffer F3 to the sample from step 3, mix completely by inverting the vial for 10 times. Incubate at room temperature for 2-3 min. Transfer the whole lysate to a lysate clearance column.

5. Centrifuge at 12,000 rpm for 30 seconds. Note: If the lysate still remains in the column, spin for another 30 seconds.

6. Discard the lysate clearance column and add 200 µL 100% ethanol to the flow through in the collection tube, mix well by pipetting and transfer the sample to a DNA mini column.

7. Spin at 12,000 rm for 30 seconds. Discard the flow through and use use the collection tube

8. Optional: Add 300 µL Buffer KB and centrifuge at 12,000 rpm for 30s. Discard the flow-through and put the column back to the collection tube. Note: This step is NOT necessary if the plasmid is being purified from endA- strain such as DH5a and Top 10. Buffer KB wash is necessary for endA+ strains such as HB101, JM110, JM 101 and their derived strains.

9. Add 600 µL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 12,000 rpm (14,000 - 18,000 x g) for 30 seconds at RT. Remove the spin column from the tube and discard the flow-through.

10. Reinsert the spin column into the collection tube and centrifuge for 1 minute at 13,000 rpm. Note: Residual ethanol will be removed more effectively with the column lid open.

11. Carefully transfer the spin column into an Elution tube (Provided) and add 50-100 µL Elution Buffer into the column and let it stand for 1 minute. Elute the DNA by centrifugation at 12,000 rpm (14,000-18,000 x g) for 30 seconds to elute DNA.

Note: The first elution normally yields around 70% of the plasmid DNA bound to the column. Add the eluted DNA back to the column for another elution yields 20-30% of the DNA bound to the column.


DNA Extraction and Purification

Used to isolate bacteria, fungi, plant and animal genomic DNA from soil and environmental samples. DNA is purified by silica-based spin filter columns and is suitable for PCR analysis and other downstream applications.

Invitrogen&trade PureLink&trade Viral RNA/DNA Mini Kit

Specifically designed to isolate high-quality viral nucleic acids from variety of RNA and DNA viruses

Thermo Scientific&trade GeneJET Genomic DNA Purification Kit

Perform silica membrane-based genomic DNA purification from mammalian cell culture and tissue samples, whole blood, bacteria and yeast.

MP Biomedicals&trade FastDNA&trade SPIN Kit for Soil

Isolates DNA from cells present in soil or other environmental samples. MP Biomedicals&trade FastDNA&trade SPIN Kit for Soil extracts DNA easily in a 2mL tube.

MP Biomedicals&trade FastDNA&trade SPIN DNA Isolation Kit for Feces

Isolates PCR-ready genomic DNA from fresh or frozen human and animal stool samples

Thermo Scientific&trade GeneJET Plasmid Miniprep Kit

Perform isolation of high quality plasmid DNA from recombinant E. coli cultures with this simple, rapid, and cost-effective system.

MP Biomedicals&trade FastDNA&trade Kit

Quickly and efficiently isolates high quality genomic DNA from plants, animals, bacteria, yeast, algae, and fungi

Invitrogen&trade UltraPure&trade TBE Buffer, 10X

Compatible with agarose gels

Thermo Scientific&trade GeneJET Plasmid Midiprep Kit

Perform large-scale isolation of high-quality plasmid DNA from recombinant E. coli cultures with this kit.

Invitrogen&trade Novex&trade Hi-Density TBE Sample Buffer (5X)

Compatible with polyacrylamide gels

MP Biomedicals&trade SPIN Module and Recovery Tubes

Designed for optimal performance, providing the purest DNA faster than ever, and with no matrix carry-over. SPIN Modules are applied in molecular biology for its ability to separate particles from solutions in a few seconds.

Qiagen Uni-Core Punch

Disposable punch for use with FTA cards, FTA Elute Cards, and EasiCollect device

Thermo Scientific&trade Glycogen, molecular biology grade

Increase the recovery of nucleic acids during preciptation with this inert carrier.


The chromatosome, a nucleosome bound to a histone H1, is the structural unit of metazoan chromatin. Determination of the high-resolution structure of the chromatosome is challenging due to the dynamic nature of H1 binding. Here, we present a protocol for purifying an optimized single-chain antibody variable fragment (scFv) that can be used to stabilize the chromatosome for single-particle cryo-EM studies. This protocol facilitates high-resolution cryo-EM structure determination of nucleosomes with a natural DNA sequence, chromatosomes, and other protein nucleosome complexes.

For complete details on the use and execution of this protocol, please refer to Zhou et al. (2021).


Biomiga Express Plasmid Mini Prep, 250 Preps (25min)

Done shopping? You can create a PDF of your cart for later or for your purchasing dept! Details at checkout.

This helpful video explains the entire process of the PD1218-02 plasmid prep kit: https://vimeo.com/231585099

This express plasmid mini prep kit includes:

Buffer F1, Buffer F2, Buffer F3, DNA Wash Buffer, Buffer KB, Elution Buffer, Lysate Clearance Column, Mini Column and RNase A

Instructions:

1. Harvest 1-2 mL of fresh bacterial culture by centrifugation for 1 minute at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely. 2. 2. Add 200 µL Buffer F1 and completely resuspend bacterial pellet by vortexing or pipetting (Complete resuspension is critical for optimal yields).

3. Add 200 µL Buffer F2, mix by inverting 10 times (do not vortex) and incubate at room temperature for 2 minutes until the solution becomes clear.

4. Add 200 µL Buffer F3 to the sample from step 3, mix completely by inverting the vial for 10 times. Incubate at room temperature for 2-3 min. Transfer the whole lysate to a lysate clearance column.

5. Centrifuge at 12,000 rpm for 30 seconds. Note: If the lysate still remains in the column, spin for another 30 seconds.

6. Discard the lysate clearance column and add 200 µL 100% ethanol to the flow through in the collection tube, mix well by pipetting and transfer the sample to a DNA mini column.

7. Spin at 12,000 rm for 30 seconds. Discard the flow through and use use the collection tube

8. Optional: Add 300 µL Buffer KB and centrifuge at 12,000 rpm for 30s. Discard the flow-through and put the column back to the collection tube. Note: This step is NOT necessary if the plasmid is being purified from endA- strain such as DH5a and Top 10. Buffer KB wash is necessary for endA+ strains such as HB101, JM110, JM 101 and their derived strains.

9. Add 600 µL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 12,000 rpm (14,000 - 18,000 x g) for 30 seconds at RT. Remove the spin column from the tube and discard the flow-through.

10. Reinsert the spin column into the collection tube and centrifuge for 1 minute at 13,000 rpm. Note: Residual ethanol will be removed more effectively with the column lid open.

11. Carefully transfer the spin column into an Elution tube (Provided) and add 50-100 µL Elution Buffer into the column and let it stand for 1 minute. Elute the DNA by centrifugation at 12,000 rpm (14,000-18,000 x g) for 30 seconds to elute DNA.

Note: The first elution normally yields around 70% of the plasmid DNA bound to the column. Add the eluted DNA back to the column for another elution yields 20-30% of the DNA bound to the column.


CONCLUSIONS

The proposed method allows students to conduct a hands-on activity focusing on the extraction and analysis of nucleic acids from different tissues, which is amenable to any secondary school classroom as a first approach to the work carried out at a molecular biology laboratory. The protocol adheres to a high safety standard at a very low cost. All of the materials used can be obtained in fact from nonspecialized suppliers. Furthermore, distinct aspects of the electrophoretic technique are intended to be addressed from an interdisciplinary stand-point, which would foster the collaborative work of teachers lecturing different courses of the secondary school science syllabi. Overall, as part of a series of lectures, combining paper modeling, animations, and traditional explanations of related recombinant DNA techniques, the protocol serves the purpose of introducing secondary school students to a research scenario in the molecular biology and genetics fields.

Additionally, our results indicate that the use of borate buffer and agar-agar gels suits many of the experiments included in college-level laboratory activities, which currently make use of more expensive agarose gels and TBE or TAE buffers. Further analysis will be performed in order to determine whether these inexpensive materials are adaptable to other applications such as recovery of nucleic acids from the gels for further manipulation or transfer to membranes for hybridization.

Home-made apparatus for horizontal gel electrophoresis. Side view (a) and top view (b) of the device, which is connected to a 12 V/500 mA adaptor used as power source. The buffer reservoirs are made from rectangular plastic food containers, and the gel casting tray and platform that connects them is cut out from a plastic soap dish. The parts were assembled and glued with hot silicone. Note that the electrodes are placed on the side of the reservoirs immediately adjacent to the gel platform. The comb is cut out from styrofoam. As shown in c, samples are loaded using 1-ml insulin syringes without the needle. A scheme of an electrical circuit (d) representing each element of the system (b) is shown (modified from Ref. 9 ).

Electrophoresis of pre-stained nucleic acids on agar-agar borate gel. Samples loaded as follows: nucleic acids extracted from kiwi (lanes 1, 4, 7, and 10), plasmid DNA (lanes 2, 5, 8, and 11), and stain alone (lanes 3, 6, 9, and 12). The stains shown are: Victoria Blue (lanes 1–3), Safranin O (lanes 4–6), Crystal Violet (lanes 7–9), and Methylene Blue (lanes 10–12). The relative position of the electrodes is shown. The gels were photographed using a reflex photo camera with macro lens (and contrasted digitally using Adobe Photoshop 7.0). The gels were run for 1 h on home-made chambers at 12 V.

Analysis of the protocol of detection of nucleic acids. Serial dilutions of a pGEM plasmid mini-preparation (not treated with RNase), pre-stained as described in the text with 0.075 g/liter Methylene Blue, were loaded on a 0.8% agar-agar borate gel and electrophoresed at 12 V for 1 h. After being photographed with a reflex photo camera (a), the gel was submerged in a 0.5-μg/ml ethidium bromide solution for 20 min with gentle shaking and photographed under ultraviolet light (b). The actual quantity of nucleic acids loaded (as determined spectrophotometrically for each sample and according to the supplier for the ladder) is as follows: lane 1, 1-kb ladder (New England Biolabs, Beverly, MA), 2.5 μg lane 2, Methylene Blue, no nucleic acids lane 3, 60 μg lane 4, 6 μg lane 5, 1 μg lane 6, 0.5 μg. The colored bands in lane 1 (a) correspond to bromphenol blue and Xylene Cyanol Blue of the loading buffer of the ladder. The photograph in a was contrasted digitally using Adobe Photoshop 7.0.

Proposed pedagogical setting: the obtention of the sequence of the human Genome. The experimental protocol described in the article is used to mimic the first stage of this scientific enterprise. See text for further details.

1.5% sodium chloride-10% kitchen detergent solution in tap water (750 ml)

coffee filters or filter paper (6)

glass or plastic containers or beakers (6)

alcohol (ethanol or isopropanol) (150 ml)

evaporating dish or small shallow plastic or glass container (30 alternatively, the bottom cut out from plastic bottles can be used for this end).

basic stain (Methylene blue, Crystal violet, Victoria Blue, Safranin O) (25 ml of a 0.075 g/liter aqueous solution in 20% glycerol)

borate buffer, 3.8 g/liter (approximately 6 liter, depending on the size of the electrophoresis chambers)

agar-agar (approximately 5 g)

electrophoretic chambers (6)

12 V AC/DC adaptors (500 mA max) (6)

insulin syringes without needle, dropper or Pasteur pipettes (glass or plastic) (30)

scanner, camera, light-box, or overhead projector

Extraction of nucleic acids and preparation of samples for electrophoresis (45–60 min). The samples are stored in the freezer (or refrigerator) until next session.

Gel casting and discussion of the electrophoretic system properties and functioning (measure of voltage, observation of reactions on the electrodes) (1 h). The gel may be stored in the refrigerator or at room temperature overlaid with buffer.

Electrophoretic run, documentation, and discussion of results (2 h). The time allowed for sample migration can be used for other activities or to finish the discussion of results from the previous sessions.

Make sure the plastic bag used in step 1 is resistant.

A blender or mortar can be used in place of the plastic bag.

Be careful not to puncture the filter. If fruit pieces are detected on the filtrate, repeat the procedure with a new filter.

The disruption and filtration steps should be performed as fast as possible, to avoid the action of nucleases.

In some countries or states, regulations do not permit the use of ethanol in schools. Isopropanol (rubbing alcohol) can be used instead for nucleic acids precipitation.

Although the present protocol indicates the use of room temperature alcohol (approximately 20°C), most protocols advise the use of ice-cold alcohol in order to maximize yield. If you should obtain no precipitate in this step, try this modification.

Spoolable DNA is usually obtained from kiwi. In any case, the nucleic acids precipitate can be recovered from the alcoholic phase by sliding it over the walls of the tube into the evaporating dish, with the aid of the rod. Inoculation loops also work very well for this purpose.

The same kind of paper used to filter the solution can be used to eliminate the alcohol from the pellet placed in the evaporating dish.

The precipitate should not be allowed to dry completely because this would make its resuspension more difficult. Generally, air-drying for 10 min is enough.

Glycerol is used to increase the density of the nucleic acids preparation for its loading in the gel. A concentrated solution of sucrose can be used in its place.

Distilled water may be used for reagents preparation if available, but is only necessary when the quality of the tap water is not adequate.

It is convenient to prepare stock solutions of the different stains (at a 5 g/liter concentration, for instance). In order to prepare the working solutions of Methylene Blue, Crystal Violet, and Safranin O, add 1 ml of the corresponding 5 g/liter stock solution (which can be measured with an insulin syringe or calibrated dropper pipette) to 66 ml of 20% glycerol aqueous solution. For Victoria Blue B, add 17 ml of the 5 g/liter stock solution to 83 ml of 20% glycerol aqueous solution. Stock solutions should be stored in dark glass bottles.

Staining may display variability depending on the age of the stain powders used and on the storage time of stock solutions.

The pre-stained samples may be stored in the freezer until their electrophoretic analysis.

Make sure to use an uncapped or loosely capped microwave-resistant bottle (or Erlenmeyer) to heat the agar-agar solution.

Usually, 2 min in the microwave at medium power is enough to dissolve 0.8 g of agar-agar in 100 ml of borate buffer (note that this should be adjusted for each microwave). Monitor this step to avoid spillages. Make sure the agar-agar is dissolved by carefully swirling the bottle and checking for visible particles. If needed, increase heating time.

Be careful not to burn yourself when handling the hot solution and do not place the bottle on cold surfaces.

Allow the solution to cool off until the bottle can be held with the hands, before pouring it into the casting tray.

If a microwave is unavailable, the solution can be heated in a boiling water bath.

A dark-colored tape can be placed on the bottom of the casting tray just below the comb, in order to see the wells more easily during the loading of the samples.

Most protocols for nucleic acids electrophoresis place the comb closer to the cathode. However, in the proposed protocol is important to place it in the middle of the casting tray (see “Discussion”).

Make sure that the comb does not move when pouring the agar solution and during the solidification process.

The comb should be removed very carefully as the styrofoam may adhere to the gel and tear the wells.

The gel casting tray can be placed in the refrigerator after pouring the agar-agar in order to accelerate its hardening.

Gel concentration can be reduced to 0.4% in order to use less agar-agar. In this case, extra care should be taken to remove the comb.

Both the agar-agar and the borate buffer can be reused after the run.

Medicine droppers bought at a drugstore or supermarket can also be used for sample loading.

If the nucleic acids preparation is not adequately resuspended, the sample might get out of the well when loaded or fail to enter the gel, once the electrical current is connected. If this should happen, add more staining solution to the sample, mix thoroughly and repeat the loading step.

It may be useful to determine the actual electrical field achieved across the gel in each particular electrophoretic system. To do so, measure the ΔV by placing each test lead of a voltmeter touching opposing ends of the submerged gel and divide the resulting value by the length of the gel. Reducing the distance between the chamber electrodes will result in a grater field. In addition, the volume of the electrolyte solution overlying the gel can be reduced to maximize the electrical current flowing through the gel (see Fig. 1 and “Discussion”).

When shorter running times are required, a variable voltage power source can be used in place of the 12-V adaptor. In this case, due precautions should considered to minimize the electrical hazard. Also, due to the electrostatic nature of the interactions between the basic stains and the nucleic acids, high voltages might not be compatible with this protocol.

If copper electrodes are used, the anode should be cleaned periodically during the run, as the Cu(OH) 2 precipitate may interfere with the electrical flux. Also, keep in mind that copper wire is usually sold with an insulating plastic covering (generally transparent), which should be removed by flaming the wire. Alternatively, stainless-steel wire can be used.

If significant voltage or pH variations are detected in the course of the run, the buffer reservoirs of the constructed chamber may be too small.

When using Methylene Blue, Crystal Violet, or Safranin O as stains, results should be documented as soon as possible (e.g. within an hour of the end of the run), because of their rapid diffusion. On the other hand, gels of nucleic acids stained with Victoria Blue B can be stored in a refrigerator for several days with no significant loss of staining.


Methods for Determining DNA Yield & Purity by Absorbance

DNA yield can be assessed using three different physical methods: absorbance (optical density), agarose gel electrophoresis and fluorescent DNA-binding dyes. Each technique is described below and includes information on necessary accessories (e.g., equipment). While all methods are useful, each has caveats to consider when choosing a quantitation approach.

The most common technique to determine DNA yield and purity is also the easiest method—absorbance. All that is needed for measurement is a spectrophotometer equipped with a UV lamp, UV-transparent cuvettes (depending on the instrument) and a solution of purified DNA. Absorbance readings are performed at 260nm (A260) where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution. To ensure the numbers are useful, the A260 reading should be between 0.1–1.0.

However, DNA is not the only molecule that can absorb UV light at 260nm. Since RNA also has a great absorbance at 260nm, and the aromatic amino acids present in protein absorb at 280nm, both contaminants, if present in the DNA solution, will contribute to the total measurement at 260nm. Additionally, the presence of guanidine will lead to higher 260nm absorbance. This means that if the A260 number is used for calculation of yield, the DNA quantity may be overestimated (43).

To evaluate DNA purity by spectrophotometry, measure absorbance from 230nm to 320nm in order to detect other possible contaminants present in the DNA solution. The most common purity calculation is determining the ratio of the absorbance at 260nm divided by the reading at 280nm. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present. However, the best test of DNA quality is functionality in the application of interest (e.g., real-time PCR).

Strong absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260nm to 230nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. As a guideline, the A260/A230 is best if greater than 1.5. A reading at 320nm will indicate if there is turbidity in the solution, another indication of possible contamination. Therefore, taking a spectrum of readings from 230nm to 320nm is most informative.

Agarose gel electrophoresis of the purified DNA eliminates some of the issues associated with absorbance readings. To use this method, a horizontal gel electrophoresis tank with an external power supply, analytical-grade agarose, an appropriate running buffer (e.g., 1X TAE) and an intercalating DNA dye along with appropriately sized DNA standards are needed for quantitation. A sample of the isolated DNA is loaded into a well of the agarose gel and then exposed to an electric field. The negatively charged DNA backbone migrates toward the anode. Since small DNA fragments migrate faster, the DNA is separated by size. The percentage of agarose in the gel will determine what size range of DNA will be resolved with the greatest clarity (40). Any RNA, nucleotides and protein in the sample migrate at different rates compared to the DNA so the band(s) containing the DNA will be distinct.

Concentration and yield can be determined after gel electrophoresis is completed by comparing the sample DNA intensity to that of a DNA quantitation standard. For example, if a 2µl sample of undiluted DNA loaded on the gel has the same approximate intensity as the 100ng standard, then the solution concentration is 50ng/µl (100ng divided by 2µl). Standards used for quantitation should be labeled as such and be the same size as the sample DNA being analyzed. In order to visualize the DNA in the agarose gel, staining with an intercalating dye such as ethidium bromide or SYBR® Green is required. Because ethidium bromide is a known mutagen, precautions need to be taken for its proper use and disposal (43).

DNA-binding dyes compare the unknown sample to a standard curve of DNA, but genomic, fragment and plasmid DNA will each require their own standard curves and cannot be used interchangeably. If the DNA sample has been diluted, you will need to account for the dilution factor when calculating final concentration. Hoechst bisbenzimidazole dyes or PicoGreen® selectively bind double-stranded DNA (dsDNA). To use this method, a fluorometer to detect the dyes, dilution of the DNA solution and appropriate DNA standards are required. However, there are size qualifications: the DNA needs to be at least 1 kilobase in length for Hoechst and at least 200bp for PicoGreen® for successful quantitation. The range of measurement is 10–250ng/ml for Hoechst, 25pg/ml–1µg/ml for PicoGreen®, and the dyes are sensitive to GC content. In addition, the usual caveats for handling fluorescent compounds apply—photobleaching and quenching will affect the signal. While the dyes bind preferentially to dsDNA, RNA and nucleotides may contribute to the signal.

Choosing which quantitation method to use is based on many factors including access to equipment or reagents, reliability and consistency of the concentration calculations. Use caution when comparing yields between methods as the level of potential contaminants may cause variable determinations among the different methods.

Webinar: To NanoDrop or Not to NanoDrop: Choosing the Most Appropriate Method for Nucleic Acid Quantitation

Learn about the advantages and disadvantages of current DNA/RNA quantitation methods, including absorbance, fluorescent nucleic acid-binding dyes and qPCR.


The majority of the mammalian genome is transcribed into non-coding RNAs, many of which co-evolve with RNA-binding proteins (RBPs) to function as biochemically defined and tractable ribonucleoproteins (RNPs). Here, we applied icSHAPE- a robust and versatile RNA structural probing pipeline- to endogenous RNPs purified from nuclei, providing base-resolution structural rationale for RNP activity and subcellular localization. Combining with genetic and biochemical reconstitutions, structural and functional alternations can be directly attributed to a given RBP without ambiguity.

For complete details on the use and execution of this protocol, please refer to Chen et al. (2018).


DNA Extraction and Purification

Perform isolation of high quality plasmid DNA from recombinant E. coli cultures with this simple, rapid, and cost-effective system.

Thermo Scientific&trade GeneJET Gel Extraction Kit

Perform silica membrane-based DNA fragment purification from standard or low-melting point agarose gels run in either TAE or TBE buffer.

Thermo Scientific&trade GeneJET Plasmid Maxiprep Kit

Perform large-scale isolation of high-quality plasmid DNA from recombinant E. coli cultures with this kit.

Qiagen Non-indicating FTA Classic Cards

Lyses cells on contact, denatures proteins and protects DNA from degradation. Whatman&trade Non-indicating FTA Classic Cards contain chemical denaturants and a free radical scavenger keeping DNA tightly bound while proteins and inhibitors are washed from the matrix.

Thermo Scientific&trade GeneJET Genomic DNA Purification Kit

Perform silica membrane-based genomic DNA purification from mammalian cell culture and tissue samples, whole blood, bacteria and yeast.

Cytiva illustra&trade GenomiPhi V2 DNA Amplification Kit

Amplify whole genomes with high efficiency and representation. Cytiva Life Sciences&trade illustra&trade GenomiPhi V2 DNA Amplification Kit provides whole genome amplification with yields of 4 to 7&mug.

Thermo Scientific&trade Glycogen, molecular biology grade

Increase the recovery of nucleic acids during preciptation with this inert carrier.

Thermo Scientific&trade Silica Bead DNA Gel Extraction Kit

Perform silica bead-based DNA extraction from agarose gels and reaction mixtures with this kit, which is flexible to scale up or down both reaction and elution volumes.

MP Biomedicals&trade FastDNA&trade Kit

Quickly and efficiently isolates high quality genomic DNA from plants, animals, bacteria, yeast, algae, and fungi

Cytiva illustra&trade NAP&trade Columns, NAP-5

Use for rapid and efficient purification of DNA and oligonucleotides (>10-mer) in less than 15 minutes by gravity flow. Cytiva Life Sciences&trade illustra&trade NAP&trade Columns are disposable columns prepacked with Sephadex&trade G-25 DNA Grade

Invitrogen&trade PureLink&trade PCR Micro Kit

Invitrogen&trade CloneChecker&trade System (for screening bacterial colonies)

From plated colonies through lysis in less than 5 minutes

Thermo Scientific&trade Lysis Buffer for MagJET Blood gDNA Kit

Generate sample lysate in order to isolate genomic DNA. Thermo Scientific&trade MagJET&trade Lysate Buffer is a component of the MagJET&trade Whole Blood gDNA Kits (K2741&frasl2742) and may be purchased separately when additional solution is needed. It is provided in 15mL bottles.

Thermo Scientific&trade Lysis Buffer for MagJET Plasmid DNA Kit

Generate sample lysate in order to isolate plasmid DNA. Thermo Scientific&trade MagJET&trade Lysate Buffer is a component of the MagJET&trade Plasmid DNA Kits (K2791&frasl2792) and may be purchased separately when additional solution is needed. It is provided in 25mL bottles.

Thermo Scientific&trade Wash Buffer 1 for MagJET Viral Kit (concentrated)

Wash lysed samples to remove any contaminants or inhibitors from the isolated nucleic acids. Thermo Scientific&trade MagJET&trade Wash Buffer 1 (concentrated) is a component of the MagJET&trade Viral DNA and RNA Kits (K2781&frasl2782) and may be purchased separately in 90mL aliquots.

Invitrogen&trade PureLink&trade 96 Receiver Plates

Purify 96 or 384 plasmid DNA samples simultaneously

Thermo Scientific&trade Precipitation Solution for MagJET Plant gDNA Kit

Precipitate the sample lysate in order to isolate plant genomic DNA. Thermo Scientific&trade MagJET&trade Precipitation Solution is a component of the MagJET&trade Plant gDNA Kits (K2761&frasl2762) and may be purchased separately in 16.5mL aliquots.

Invitrogen&trade PureLink&trade HiPure Plasmid Filter Maxiprep Kit

Purification in 90 minutes

Thermo Scientific&trade Elution Buffer for MagJET Plasmid DNA Kit

Elute purified plasmid DNA for use in common molecular biology procedures. Thermo Scientific&trade MagJET&trade Elution Buffer is a component of the MagJET&trade Plasmid DNA Kits (K2791/2792) and may be purchased separately when additional solution is needed. It is provided in 18mL aliquots.


RNA Isolation

This is a continuation of the procedures from Sample Preparation in Section A, B, C and D.

1. Load up to 700 μl of lysate/ethanol mixture into RNA Spin Column placed in a 2ml Collect Tube, spin at top

speed (> 10,000 rpm) for 1 min, and discard the flow-through.

* If lysate/ethanol mixture is greater than 700μl, apply any remaining lysate/ethanol into the column and repeat

centrifuge step as above.

2. Returning the Column to the original Collection Tube, and add 500 μl RNA Wash Buffer I to the column,

(> 10,000 rpm) for 1 min, and discard the flow-through.

3. For each sample, mix 80 μl DNase Incubation Buffer with 2 μl DNase I in another centrifuge tube (Mix by

flicking or inverting the tube, do not vortex!). Pipet the solution into the center of spin column, and incubate at

* If multiple sample are performed, prepare master mix of DNase I solution just before loading into the column,

do not store premix DNase I solution.

4. Add 500 μl RNA Wash buffer I to the column, spin at top speed (> 10,000 rpm) for 1 min, and discard the

flow-through and Collect Tube.

5. Combine the Spin Column with a new Collect Tube, add 600 μl RNA Wash Buffer II to the column, spin at

top speed (> 10,000 rpm) for 1 min, and discard the flow-through.

6. Add another 600 μl RNA Wash Solution II to the column, spin at top speed (> 10,000 rpm) for 1 min, and

7. Returning the Spin Column with original Collect Tube and spin at top speed (> 10,000 rpm) for 3 min to remove

* If centrifuge speed is lower than 10,000 rpm or residual ethanol must be removed completely, incubate the spin

column at 45-60°C oven for 5 min to evaporate all of the ethanol.

8. Add 30-50 μl nuclease-free water to the center of membrane, and centrifuge for 1 min at top speed. Store the

Appendix I: Protocol for isolation of total RNA from heart, muscle and skin tissues

Additional reagent and equipment required:

1. Process and homogenize the sample as Sample Preparation Section B Animal Tissue procedure (I) and

2. Incubate at 56°C for 10 min.

3. Spin the lysate at top speed for 3 min, carefully transfer the supernatant to a new tube without disturbing the pellet.

4. Add 0.5 volume of 100% ethanol to the lysate, mix by pipetting. Continue to RNA Isolation Section.

* Do not centrifuge after adding ethanol.

Apprendix II: Protocol for isolation of total RNA from white blood cells of fresh blood sample

Additional reagent and equipment required:

clear red) invert the tube 2-3 times during incubation.

2. Centrifuge for 3,500 x g for 5 min, remove supernatant without disturbing the pellet.

3. Resuspend and wash the leukocyte pellet in 2 volumes of 1X RBC Lysis Buffer. Mix well by vortexing briefly.

4. Centrifuge for 3,500 x g for 5 min, remove supernatant without disturbing the pellet.

5. Vortex or flicking the tube to loosen cells, add 350 μl of RNA Lysis/2-ME Buffer to the cells.

6. Vortex or pipette thoroughly until clump disappear.

7. Homogenization cells for 30 s by rotor-stator or pass the lysate through a 20G needle for at least 5-10 times,

or until a homogenous lysate without sticky appearance.

8. Add350 μl of 70% Ethanol to the lysate, and mix well by vortex or pipetting. Continue to RNA Isolation

* Do not centrifuge after adding ethanol.

Apprendix III: Protocol for RNA Clean-Up or genomic DNA Removal

This kit can be used to clean up RNA or to remove genomic DNA contamination from other isolation methods.

1. Adjust RNA volume to 100 μl with nuclease-free water. Add 350 μl of RNA Lysis/2-ME Buffer and mix well.