Nucleic acid detection/staining - Info

Nucleic acid detection/staining

CHOOSE THE RIGHT STAIN FOR YOUR APPLICATION

Product	Method	Procedure	Advantages	Disadvantages	Recommended for
DNA prestaining	GelRed® Prestain Plus 6X DNA Loading Dye	GelRed® loading buffer is added directly to the DNA sample before loading	Fast & simple: one-step sample loading & DNA staining Less concentrated dye for safer handling Can re-run a gel to use empty lanes	Not recommended for PAGE, DGGE, EMSA, or PFGE gels Dye may cause band migration issues when loading larger amounts of DNA (more than ~100 ng/band), or for some restriction digests	Routine agarose gels Recommended loading 50-200 ng ladder or 2-5 uL PCR product ( ~100 ng/band or less)
Precast staining	GelRed® 10,000X in water or GelGreen® 10,000X in water	GelRed® or GelGreen® is mixed with molten agarose before gel casting	Familiar protocol, rapid results
Precast staining	GelRed® Agarose LE or GelGreen® Agarose LE	Agarose is supplied pre-coated with GelRed® or GelGreen®, just dissolve, heat, and pour	Safer & more convenient, no need to handle concentrated dye
Post-electrophoresis staining	GelRed® 10,000X in water or GelGreen® 10,000X in water – or – GelRed® 3X in water	No fluorescent dye is added to the gel, it is stained in 3X GelRed® or 3X GelGreen® solution after electrophoresis	Most accurate sizing/sharpest bands Staining solution can be re-used Enhance sensitivity by adding NaCl	Extra staining step (up to 30 minutes) after electrophoresis (some customers report good results after only 5 minutes if dye is not reused)	Highly accurate band sizing Gels with more than ~100 ng DNA per band Analyzing restriction digests
Post-electrophoresis staining of PAGE Gels	PAGE GelRed® 10,000X or 1X in water	No fluorescent dye is added to the gel, it is stained in 1X PAGE GelRed® solution after electrophoresis	Formulated for efficient penetration and staining of polyacrylamide gels Like the classic GelRed®, it is safe and environmentally friendly	Extra staining step of approx. 30 minutes after electrophoresis	Staining of nucleic acids in PAGE gels

Products

GelRed™ and GelGreen™ - DNA Stains Biotium

Convenient for the Lab – environmentally friendly

To visualize nucleic acids the experimenter makes use of color stains that interact with them. One of those stains is ethidium bromide (EtBr) which can be illustrated in an agarose or polyacrylamide gel when exposed to UV rays. EtBr is proven to be a highly toxic, cancerous and mutagenic substance which calls for cautious handling. It is linked to frameshifting in the DNA which leads to mutation and therefore a loss of function in proteins. To minimize factors of possible damages that are accompanied with the use of EtBr other substances like SYBR Green I + II, OliGreen or RedSafe which are said to be less harmful can be used.

Biotium offers two alternatives to EtBr which do not penetrate neither latex gloves nor the cell membrane and therefore cannot interact with the DNA - GelRed™ and GelGreen™. Additionally a higher sensitivity for better quantification and less sample use can be achieved.

Mechanism of DNA Intercalation of ethidium bromide

Figure 1 ethidium bromide (red bars) settles between base pairs and alternates the structure. This leads to a shift in reading frame of the DNA. For visualization of DNA essential. Instead of EtBr you can use GelRed™ and GelGreen™.

Differences of GelRed™ and GelGreen™ to common stains (ethidium bromide, SYBR)

1. Do not penetrate Latex gloves.

a. Glove Penetration Test

2. Do not penetrate cell membrane.

a. Cell staining test

3.Biodegradable.

a. Can be disposed down the drain or in regular trash.

4.Higher sensitivity.

Figure 2 Comparison of ethidium bromide (EtBr) and GelRed in precast gel staining in 1% agarose/TBE gel. Two-fold serial dilutions of 1 kb Plus DNA Ladder (Invitrogen) were loaded in the amounts of 200 ng, 100 ng, 50 ng and 25 ng from left to right. Gels were imaged using 300 nm transilluminator and photographed with an EtBr filter.

Figure 3 Comparison of GelGreen and SYBR Safe in post-electrophoresis staining of 1% agarose/TBE gels. Two-fold serial dilutions of 1 kb Plus DNA Ladder (Invitrogen) were loaded in the amounts of 200 ng, 100 ng, 50 ng and 25 ng from left to right. Gels were imaged using 254-nm transilluminator and photographed with a SYBR filter.

For more information read the Safety Report of Biotium.

Literature:

(1) Mülhardt, Der Experimentator – Molekularbiologie Genomics, K.3 S. 62

Image register

(1) Mechanism of ethidium bromide intercalation into DNA. Created with ChemSketch and Photoshop

(2) https://biotium.com/wp-content/uploads/2013/09/41003-EtBr-vs-GelRed-labeled-wider-266x300.jpg Status: 29.03.2016 15:25

(3) https://biotium.com/wp-content/uploads/2013/09/41005-SYBRSafe-vs-GelGreen-labeled-266x300.jpg Status: 29.03.2016 15:25

Nucleic acid detection/staining - Info