3)SybrGreen is recommended for visualisation. When ethidium bromide is used for visualisation the addition of ethidium bromide to the electrophoresis buffer (0.01µg/ml) improves results. Nucleic acid loading buffer must be prepared using RNase free reagents/solutions.

4) The integrity of the RNA may be assessed by the absence of smearing and the fluorescent signal, the ratio of 28S to 18S RNA should be 2:1.

5) 10x SSC or 20x SCC can be used as the transfer buffer.

Glyoxal protocol

1) Prepare the MOPS gel as follows: Preheat 30ml 10x MOPS buffer at 55°C. Dissolve 3 - 4.5g of agarose in 270ml of nuclease free water. Cool to 55°C. Add the 10x MOPS buffer. Cast the gel in an appropriate enclosure and allow the gel to set.

 2)Prepare the RNA sample(s), using the table below:

Place the sample(s) at 50°C for 60 minutes to denature. After denaturation add 3µl of 10x nucleic acid dye loading buffer. Mix and load onto the agarose gel.

3)Separate the RNA samples using lx MOPS buffer as the electrophoresis buffer.

4) Following electrophoresis, if appropriate, visualise the RNA within the gel with UV light and photograph.

5) Set up the capillary blot as described using a neutral transfer buffer.

Notes

1) Glyoxal oxidizes very rapidly. Stock solutions 40%(w/v) or 6M must be deionized to neutral pH before use. Small aliquots can then be stored at -20°C in tightly capped tubes. Once thawed use only once.

2) Glyoxylated RNA should be run more slowly than formaldehyde gel to prevent formation of pH gradients.

3) Glyoxal gels may be electrophoresed in 1x MOPS thereby eliminating the need to recirculate the buffer.

4) Glyoxal may interact with ethidium bromide altering the dyes spectral properties.

5) No pre treatment of the gel is required. Glyoxal is removed from the RNA during prehybridization or any post fixation washes of the blot.

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