|SYBR Gold is the most sensitive of the dyes we have tested for the direct visual detection of dsDNA and it is possible to see less than 100 pg of dsDNA by eye on a Dark Reader transilluminator. Using a CCD camera or Polaroid film it is possible to detect 10 - 25 pg.
Though we have not tried it ourselves yet, we have had several reports from DR users that SYBR Gold also works well with RNA and ssDNA.
|A 2-fold dilution series of lambda DNA cut with StyI/SauI (Roche mol. weight marker IV) was subjected to electrophoresis in 1% SeaKem LE agarose (Lonza Bioscience) in 1 x TAE buffer for 50 minutes at 5 V/cm. The total amount of DNA loaded per lane ranged from 12.5 ng down to 0.39 ng. The gel was subsequently stained with a 1:10,000 dilution of SYBR Gold stain in 1 x TAE for 30 minutes and photographed on a DR190M using an Olympus C3000 digital camera.|
Tips on using the new DNA stains
|Here are a few tips on usage of SYBR Gold. (Most of these tips also apply to SYBR Green, and GelStar too.)
1. Because it can take 15 - 20 minutes for the 0.5 mL stock DMSO solution to thaw, we normally divide the stock solution up into 10 uL aliquots before storing at -20 oC. This reduces the thaw time to a few seconds and each aliquot is convenient for making a 100 mL of stain solution.
2. Using SYBR Gold with the Dark Reader, we obtain a sensitivity of dsDNA detection of about 100 pg by eye, 75 pg on Polaroid 667 film and 25 pg or less using a CCD camera system. (You will, of course, need to be in a well darkened room to see these tiny amounts of DNA by eye.)
Exposure times required for SYBR Gold-stained gels are about 2 - 10 sec using Polaroid 667 film and an f- stop of 5.6. Using a CCD camera, 2 - 4 sec is optimal, though this will depend on exactly what brand of camera you have. (Note: Do not forget to remove any non-DR filter from the camera!)
3. The agarose and gel buffers should be reasonably fresh and free of debris as this can markedly increase the background fluorescence from the gel itself and mask the DNA fluorescence (SYBR Green seems to suffer less from this problem). Also, keep the thickness of the gel to a minimum to reduce background fluorescence.
4. We have had a few complaints that SYBR Gold is TOO sensitive! If your DNA sample contains a lot of `heterogeneity` then, because of the sensitivity of SYBR Gold, a visible smearing may occur that masks the bands of interest. To record a usable image of the gel, the camera exposure time should be reduced to such an extent that the smearing is faded.
The alternative (and better) approach is to reduce the amount of DNA loaded onto the gel. This also results in significant cost savings in terms of the smaller amounts of DNA standards and PCR reaction mixes that are required on a gel. (See link to right for a detailed analysis.)
5. We have occasionally noticed a peculiar transient smearing effect. This becomes visible after about 5 minutes or so of staining time but disappears within 30 minutes. We have no idea what causes this.)
6. SYBR Gold enters the agarose very rapidly. It is possible to see major bands within a couple of minutes. (To quickly check on the extent of staining it is not necessary to remove the gel from the staining dish. We typically use the semi-transparent lids of pipette tip boxes which the DR light passes through fairly well.)
7. Unfortunately, the SYBRs are not very stable in aqueous solution (The exception is SYBR Safe). We typically keep a staining solution around for a couple of weeks at 4 oC.
8. We have not had any success using SYBR Gold as a pre-stain for dsDNA samples as it severely retards DNA migration. However, a recent report (McKee & Thomson, BioTechniques 37: 46-50, 2004) describes using SYBR Gold as a pre-stain in an SSCP protocol.
Clare Chemical Research