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Improving Gel Extraction yields

Improving Gel Extraction yields


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How can I improve my Gel Extraction yields. We use the standard protocol from Qiagen, gel extraction, dissolve in QG buffer at 42C and purify via anion exchange columns. However, with 500 ng we ultimately get 100 ng for a 20% yield. I was curious what else has been tried for gel extraction.

We also add IPA and NaOAc to the dissolved mixture prior to the column purification.


When I optimized gel extraction in my hands, two factors turned out to be critical in maximizing gel extraction yield:

The pH of the eluting solution. I used to elute the final product with DEPC-treated water initially, but the yield would vary a lot as the pH of the water changed. Using buffer EB is better, since it maintains a stable pH 8.

Most importantly, *incubate the eluting solution with the column as long a possible (and also elute twice). * I incubate for about 10 min, which is longer than what they recommend but it works much better.


Try dissolving at 50C. In the Qiagen gel extraction kit, it says to dissolve at 50C or until completely dissolved.

The size of the gel is important too. Make sure it is 1.5% or less, and the size of the gel needs to be less than 400 mg.

Also, pay attention to the first step. If the liquid is not the same color as the color of the first QG buffer then there is a pH problem.


Elute from the column with Elution Buffer that has been preheated to 50-60 degrees. I stick mine in the incubator for the duration of the extraction so it is warm when I come to elute. It seems to help yield.


Techniques for extraction and isolation of natural products: a comprehensive review

Natural medicines were the only option for the prevention and treatment of human diseases for thousands of years. Natural products are important sources for drug development. The amounts of bioactive natural products in natural medicines are always fairly low. Today, it is very crucial to develop effective and selective methods for the extraction and isolation of those bioactive natural products. This paper intends to provide a comprehensive view of a variety of methods used in the extraction and isolation of natural products. This paper also presents the advantage, disadvantage and practical examples of conventional and modern techniques involved in natural products research.


Monarch® DNA Gel Extraction Kit

Quickly and easily purify DNA from agarose gels with high yields.

  • Elute in as little as 6 &mul
  • Prevent buffer retention and salt carry-over with optimized column design
  • Save time with fast, user-friendly protocols
  • No need to monitor pH or add isopropanol
  • Buffers and columns available separately
  • Significantly less plastic used when compared with other kits
  • Responsibly-sourced and recyclable packaging

Check out our Technical Note containing comprehensive insights into measuring and analyzing nucleic acids.

The Monarch DNA Gel Extraction Kit rapidly and reliably purifies up to 5 &mug of concentrated, high-quality DNA from agarose gels. The kit utilizes a bind/wash/elute workflow with minimal incubation and spin times. The columns ensure zero buffer retention and no carryover of contaminants, enabling elution of sample in volumes as low as 6 &mul. The buffers provided have been optimized, and do not require monitoring of pH. Unlike other kits, there is no need to add isopropanol to the melted agarose prior to loading on the column, saving you a step. Eluted DNA is ready for use in restriction digests, DNA sequencing, ligation and other enzymatic manipulations. Designed with sustainability in mind, Monarch kits use significantly less plastic and responsibly-sourced, recyclable packaging


View our videos on protocols, tips, and recycling Monarch.

  • Elute in as little as 6 &mul
  • Prevent buffer retention and salt carry-over with optimized column design
  • Save time with fast, user-friendly protocols
  • No need to monitor pH or add isopropanol
  • Buffers and columns available separately
  • Significantly less plastic used when compared with other kits
  • Responsibly-sourced and recyclable packaging

Kit Components

The following reagents are supplied with this product:

Monarch® DNA Cleanup Columns (5 &mug) T1034-2 25 1 x 50 columns Not Applicable
Monarch Collection Tubes (2ml) T1018-2 25 1 x 50 tubes Not Applicable
Monarch® DNA Wash Buffer T1032-2 25 1 x 5 ml Not Applicable
Monarch® Gel Dissolving Buffer T1021-2 25 1 x 47 ml Not Applicable
Monarch® DNA Elution Buffer T1016-21 25 1 x 3 ml Not Applicable
Monarch® DNA Cleanup Columns (5 &mug) T1034-2 25 5 x 50 columns Not Applicable
Monarch Collection Tubes (2ml) T1018-2 25 5 x 50 tubes Not Applicable
Monarch® DNA Wash Buffer T1032-3 25 1 x 25 ml Not Applicable
Monarch® Gel Dissolving Buffer T1021-3 25 1 x 235 ml Not Applicable
Monarch® DNA Elution Buffer T1016-2 25 1 x 7 ml Not Applicable

Features

  • Elute in as little as 6 &mul
  • Prevent buffer retention and salt carry-over with optimized column design
  • Save time with fast, user-friendly protocols
  • No need to monitor pH or add isopropanol
  • Buffers and columns available separately
  • Significantly less plastic used when compared with other kits
  • Responsibly-sourced and recyclable packaging

Companion Products

Materials Sold Separately

  1. The kit should be stored at room temperature. Always keep buffer bottles tightly closed and keep columns sealed in the enclosed zip-lock bag. For information regarding the composition of buffers, please consult the Safety Data Sheets. Proper laboratory safety practices should be employed, including the use of lab coats, gloves and eye protection.
  1. Can I purchase Monarch buffers and columns separately?
  2. Can I use water to elute the DNA when using the Monarch Kits?
  3. What is the smallest volume of elution buffer that can be used with the Monarch DNA Cleanup Column?
  4. What factors affect my (A260/A230)?
  5. Are the columns in the Monarch PCR & DNA Cleanup Kit (5 &mug) the same as the ones in the Monarch DNA Gel Extraction Kit?
  6. What is the composition of each buffer provided with the Monarch DNA Gel Extraction Kit?
  7. What is the maximum binding capacity of the Monarch DNA Cleanup Column provided in the Monarch DNA Gel Extraction Kit?
  8. Can I excise a fragment from a gel and store it for purification at a later time?
  9. What size of DNA can be purified with the Monarch DNA Cleanup Columns?
  10. What type of agarose gels are compatible with the Monarch DNA Gel Extraction Kit?
  11. After purification, I see a faint additional band running below the expected size on a gel. What happened?
  12. Are Monarch spin columns compatible with Vacuum Manifolds?
  • Reagents added incorrectly. Check protocol to ensure correct buffer reconstitution, order of addition for buffers and proper handling of column flow-through and eluents.
  • Gel slice not fully dissolved. Small clumps of agarose may clog the column or interfere with DNA binding. Be sure to incubate the gel slice in the Monarch Gel Dissolving Buffer for the specified time and within the proper temperature range. Mix the sample and inspect periodically to monitor dissolution of the agarose.
  • Gel dissolved above 60°C. The DNA may become denatured if incubated at higher temperatures than the specified range of 37&ndash55°C.
  • Incomplete elution during prep. Ensure the DNA Elution Buffer is delivered directly to the center of the column so that the matrix is completely covered and elution is efficient. Larger elution volumes and longer incubation times can increase yield of DNA off the column at the cost of dilution of the sample and increased processing times. For typical fragments below 10 kb, the recommended elution volumes and incubation times should be sufficient, unless the maximal yield is desired. For the purification of larger fragments, heating the DNA Elution Buffer to 50°C prior to eluting and extending the incubation time after buffer addition to 5 minutes can improve yield. Additionally, multiple rounds of elution can be employed to increase the amount of DNA eluted, at the expense of dilution of the sample.
  • Gel slice not fully dissolved. Undissolved agarose may leach salts into the eluted DNA. Be sure to incubate the gel slice and the Monarch Gel Dissolving Buffer mixture for the specified time and temperature. Mix the sample and inspect periodically to monitor dissolving of the agarose.
  • Ethanol has been carried over. Ensure final wash spin time is 1 minute to ensure complete removal of the wash buffer from the column and be careful when transferring the column to a new tube for elution step to ensure column tip does not contact column flow-through.
  • Trace amounts of salts that produce low OD260/230 ratios can also be carried over during the elution step. Be careful when transferring column to new tube for elution step to ensure the column tip does not contact column flow-through.
  • Troubleshooting Guide for DNA Cleanup and Plasmid Purification
Monarch DNA Gel Extraction Kit protocol
Learn how to extract DNA from agarose gels using the Monarch DNA Gel Extraction Kit. Tips for using the Monarch DNA Gel Extraction Kit
Optimize your DNA gel extractions with our quick tips for using the Monarch DNA Gel Extraction Kit.

TAE Vs TBE buffer - (Sep/11/2012 )

What's the difference between using TAE or TBE buffer for electrophoresis?

And what is the difference between using 1X TAE and 5X TAE Vs 1X TBE and 5X TBE buffer?

I use my TAE buffer (1X) for normal, shorter electrophoresis runs (PCR products, plasmid digests etc) and then TBE at 0.5X for RFLPs that I need to run overnight or longer (doesn't get as hot as the 1x TAE).

Never really questioned why, that's just how its done in my lab.

The 5X solutions will be stock solutions, that you need to dilute prior to using.

don't you use TBE buffer for running electrophoresis?

TAE is tris-acetate-EDTA, TBE is tris-borate-EDTA, they and many other buffers can be used for electrophoresis.

As leelee said TBE causes less heating because borate ions have better mobility than acetate. The problem with using borate is if you want to excise your band out of the gel and purify it the borate ions form complexes with the DNA and the yield is much lower. Therefore people use TAE. There was a paper on this subject a while ago called "Not your father's buffer" and it said that the best is Li-borate or Na-borate and in my experience you can really speed things up with a different buffer.

Thanks a lot guys.
@Biomiha - Not your father's buffer - nice article. May be i will try using Na-Borate buffer instead of TBE and TAE

I am totally for the SB buffer (better resolution) Tris based buffers are so passe

To add to the things above: you can use both for agarose electrophoresis but when I did acrylamide electrophoresis of short DNA fragments, I used TBE. More, in the case of urea denaturating gels, when the urea is not enough to obtain ssDNA, some people recommend to run everything (and prepare the gel as well) in 2X TBE to increase heating hence melting.

There was this routine to use 1x TAE gels for fragments cut out and 0.5x TBE for all other elfo, but I just smashed this tradition, because I've seen no difference in yields between the buffers (using Qiagen MinElute kit) and TAE is a worse buffer. Also not completely uninportant thing is, TAEs had to be prepared for each run from 10x solution, unlike the 0.5 TBE that we have in big tank ready to use

I'm getting more curious about the SB buffer, you all mentioned beter resolutions, possible higher running voltages and stuff, but I'm also concened about the isolation from such gels, yields, salt content (our main aim is for sequencing) stability of DNA and so on. And generaly, does it even have any disadvantages at all?

@Trof - I use SB for gel extraction and subsequent cloning and have had good yields with the Axygen gel extraction kit. I tended to get poorer yields when using TBE, but that may have been due to loading lower amounts on the gel or using a different extraction kit.

The DNA seems to be pretty stable, but I havn't assessed true integrity by mass spec or looking for crosslinking or anything else.

So far I searched the forums and made this list of SB buffer features:

pros:
- lower conductivity, less heating, higher voltage/speed possible
- better resolution (under 3 Kb)
- price (as I understand)

cons:
- lower buffering capacity, easily depleted, reuse not possible
- problems in high sample salt concentrations (possible problems in running restriction fragments?)
- not suitable over 3 kB

not sure:
gel extraction yield (particulary interested in comparison with 0.5x TBE and Qiagen kits)

Also I found more versions of protocols.
boric acid + NaOH pH 8.2
boric acid + NaOH pH 8.5
sodium tetraborate + boric acid

I'm kind of decided to do extensive testing (sharpness, resolution including very small bands, reusability, gel extraction yield) it would be better for a student but I don't have one now, but there is a new colleague that seems to be yet unoccupied so I ask if she's interested.
Our primary goal will be testing the gel extraction yields and purity, because otherwise I'm not aware about any serious resolution issues with your TBE (well didn't see anything from SB yet, maybe I'll change my mind) apart from <100 bp fragments of course, and running on higher voltage isn't really that important for us.


Tools and tech notes about cDNA synthesis.

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Conclusion

In summary, the protocol is simple and reliable, does not require grinding, centrifuging, or the use of hazardous chemicals. A large number of samples can be processed simultaneously, and full automation of the protocol is possible with existing workstations. Many different species were successfully tested. The method can be adapted to each species by modification of the digestion buffer, of the amount of the enzymatic cocktail added during the digestion or of the digestion time.

The method is perfectly adapted to situations when high-throughput isolation of PCR-ready genomic DNA is required. Moreover, because of the high-yield and high-molecular weight DNA reliably obtained, sensitive PCR-based techniques could be applied: AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), SSR (Simple Sequence Repeat polymorphism).


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DNA quantitation and purity

DNA yield and purity were determined by using Nanodrop 2000 (Thermo Scientific, Waltham, U.S.A.), a UV spectrophotometry method, at 260 nm absorption value and absorbance ratios at 260/280 nm respectively.

Primers design and qualitative PCR

Routine PCR was carried out to screen cp4epsps gene and intrinsic lectin gene of soybean. Primer pairs, Lectin-F/R 1, and Epsps-F/R 1, were used by previous studies [5,17]. Other primers were designed using software Primer Premier 5 and Oligo 7 based on genomic sequences of the lectin gene (GenBank accession: K00821) and Glycine max transgenic cp4epsps gene for EPSPS class 2 precursor (GenBank accession: AB209952). All primers were synthesized by Sangon Biotech (Shanghai, China). Sequences of primers and amplification conditions were detailed in Table 3. The conventional PCRs were performed in 25 μl total reaction volume containing 1× Premix Taq™ (TaKaRa Bio Co., Beijing, China), 0.12 μM of each primer, and 80 ng template DNA. Amplifications were carried out in TECHNE TC-412 Thermal cycler (Staffordshire, U.K.) using 5 min denaturation at 95°C initially, then 35 cycles of 30 s at 94°C, 30 s at a specific annealing temperature (Table 3), certain extension time (Table 3) at 72°C, followed by a final extension of 10 min at 72°C.

Target . Primer . Sequence (5′–3′) . Amplicon size (bp) . Annealing temperature (°C) . Extension time (s) . References .
lectinLectin-F1 GCC CTC TAC TCC ACC CCC ATC C 118 58 20 [17]
Lectin-R1 GCC CAT CTG CAA GCC TTT TTG TG
Lectin-F2 TGC CGA AGC AAC CAA ACA TGA TCC T 438 56 40 This work
Lectin-R2 TGA TGG ATC TGA TAG AAT TGA CGT T
Lectin-F3 GGC AAA CTC AGC GGA AAC TGT 772 55 60 This work
Lectin-R3 TTA GAT GGC CTC ATG CAA CAC
Lectin-F4 ACC CTT GTT AGT CAA ACC ACA 1067 55 75 This work
Lectin-R4 AAC CCT ATC CTC ACC CAC TCG
cp4epspsEpsps-F1 ATC CCA CTA TCC TTC GCA AGA 169 53 20 [5]
Epsps-R1 TGG GGT TTA TGG AAA TTG GAA
Epsps-F2 CCT TCA TGT TCG GCG GTC TCG 498 62 40 This work
Epsps-R2 GCG TCA TGA TCG GCT CGA TG
Epsps-F3 TTC ATC GGC GAC GCC TCG CTC ACA 700 64 60 This work
Epsps-R3 CGC GGA GTT CTT CCA GAC CGT TCA T
Epsps-F4 CGC CCG CAA ATC CTC TGG CCT TTC 1099 64 75 This work
Epsps-R4 CGT CTC GCC CTC ATC GCA ATC CAC
Target . Primer . Sequence (5′–3′) . Amplicon size (bp) . Annealing temperature (°C) . Extension time (s) . References .
lectinLectin-F1 GCC CTC TAC TCC ACC CCC ATC C 118 58 20 [17]
Lectin-R1 GCC CAT CTG CAA GCC TTT TTG TG
Lectin-F2 TGC CGA AGC AAC CAA ACA TGA TCC T 438 56 40 This work
Lectin-R2 TGA TGG ATC TGA TAG AAT TGA CGT T
Lectin-F3 GGC AAA CTC AGC GGA AAC TGT 772 55 60 This work
Lectin-R3 TTA GAT GGC CTC ATG CAA CAC
Lectin-F4 ACC CTT GTT AGT CAA ACC ACA 1067 55 75 This work
Lectin-R4 AAC CCT ATC CTC ACC CAC TCG
cp4epspsEpsps-F1 ATC CCA CTA TCC TTC GCA AGA 169 53 20 [5]
Epsps-R1 TGG GGT TTA TGG AAA TTG GAA
Epsps-F2 CCT TCA TGT TCG GCG GTC TCG 498 62 40 This work
Epsps-R2 GCG TCA TGA TCG GCT CGA TG
Epsps-F3 TTC ATC GGC GAC GCC TCG CTC ACA 700 64 60 This work
Epsps-R3 CGC GGA GTT CTT CCA GAC CGT TCA T
Epsps-F4 CGC CCG CAA ATC CTC TGG CCT TTC 1099 64 75 This work
Epsps-R4 CGT CTC GCC CTC ATC GCA ATC CAC

Agarose gel electrophoresis

The quality of DNA was studied by electrophoresis in a 1% agarose gel (Sangon Biotech, Shanghai, China) containing 4S Green Plus Nucleic Acid Stain (Sangon Biotech, Shanghai, China) in 1× TAE buffer. The amplification products were separated by 2% agarose gel. The agarose gel image was visualized and recorded using a UV Bio-Rad Gel Doc 2000 image detector (Bio-Rad, Hercules, U.S.A.) installing analysis software Quantity one.

Suitability test of the optimized DNA extraction method

SDS method 4 (the optimized method) was conducted to extract gDNA from Zhoudou22, Zheng196, Zhonghuang13, RRS1 and RRS2 to confirm the suitability of the optimized method.

Sensitivity of the optimized DNA extraction method

SDS method 4 (the optimized method) was applied to a range of RRS1 (100, 50, 10, 5, 1, and 0.5 mg) to extract DNA to evaluate the minimum RRS1 quantity used for DNA extraction and conventional PCR examination.

Data analysis

Each sample was tested in triplicate. One-way ANOVA analysis was conducted using SPSS software version 21.0 (IBM Analytics, Armonk, U.S.A.) to test the significance of differences amongst DNA yields.


Acknowledgements

This work was supported by the University of Queensland and a postgraduate scholarship from the Queensland Alliance for Agriculture and Food Innovation. The authors would like to thank David Lee of the University of the Sunshine Coast and the Queensland Department of Agriculture, Fisheries and Forestry (QDAFF) for providing the Corymbia leaf material for extraction providing helpful comments for the manuscript, and Jason Lupoi of the Joint BioEnergy Institute for his comments. We thank Prof. Darren Crayn of Australian Tropical Herbarium for supplying the C. brassii leaves, Myrna Constantin for endonuclease digestions, and Joint Genome Institute and Australian Genome Research Facility for library preparation and sequencing of samples.


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