RNA isolation, cDNA synthesis and rtPCR

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Analysis of PCR Products by Agarose Gel Electrophoresis

1. Dilute 5X TBE stock to 1X TBE with dH20.

2. Prepare 1.5% agarose gel for small DNA molecules (PCR products)
(Minigel apparatus holds 50 ml)

a.Dissolve agarose in 1X TBE by boiling. Allow to cool until warm (~60ºC).
Use 1 or 2 mls with pasteur pipet to seal edges of mold.

b. Add 2 ul 10 mg/ml EtBR to 50 ml agarose (wear gloves!).
Pour into mold, check for bubbles and insert comb.
BE SURE THAT COMB IS EVEN AND INTO GEL WITHOUT TOUCHING BOTTOM.

c.

Allow to harden. Remove black dams and comb. Fill tank with 1X TBE.

3. Prepare samples.
Add 10 ul loading dye to sample. Centrifuge briefly to combine under oil.
Remove 15 ul sample from under oil with gel loading pipet tip.
Load into wells.
Use 5 ul PCR markers + 5 ul dH20 + 2 ul dye.

4. Electrophorese at 80-90 volts (check that ~40 mA) until dark blue dye is approx. 1 inch from bottom.

5. Photograph using UV light box. BE SURE TO WEAR SAFETY GLASSES.

@2004 Cebra-Thomas

Last Modified: 17 August 2004

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		Analysis of PCR Products by Agarose Gel Electrophoresis 1. Dilute 5X TBE stock to 1X TBE with dH20. 2. Prepare 1.5% agarose gel for small DNA molecules (PCR products) (Minigel apparatus holds 50 ml) a.Dissolve agarose in 1X TBE by boiling. Allow to cool until warm (~60ºC). Use 1 or 2 mls with pasteur pipet to seal edges of mold. b. Add 2 ul 10 mg/ml EtBR to 50 ml agarose (wear gloves!). Pour into mold, check for bubbles and insert comb. BE SURE THAT COMB IS EVEN AND INTO GEL WITHOUT TOUCHING BOTTOM.
		c.		Allow to harden. Remove black dams and comb. Fill tank with 1X TBE.
		3. Prepare samples. Add 10 ul loading dye to sample. Centrifuge briefly to combine under oil. Remove 15 ul sample from under oil with gel loading pipet tip. Load into wells. Use 5 ul PCR markers + 5 ul dH20 + 2 ul dye. 4. Electrophorese at 80-90 volts (check that ~40 mA) until dark blue dye is approx. 1 inch from bottom. 5. Photograph using UV light box. BE SURE TO WEAR SAFETY GLASSES.