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Mycoplasma (talk | contribs) No edit summary |
Mycoplasma (talk | contribs) No edit summary |
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* Add 0.5 g (ie. 1% of 50) of agarose to 50g (or 50 ml - roughly the same) of 1X TBE. If there is no 1X TBE make it from 1 part 10X TBE (you will find in big brown bottle in bottom drawer of cabinet) and 9 parts dH20 (we use deionized water, should be on floor or in bottom drawer of cabinet). | * Add 0.5 g (ie. 1% of 50) of agarose to 50g (or 50 ml - roughly the same) of 1X TBE. If there is no 1X TBE make it from 1 part 10X TBE (you will find in big brown bottle in bottom drawer of cabinet) and 9 parts dH20 (we use deionized water, should be on floor or in bottom drawer of cabinet). | ||
* Mix and then microwave this until it boils. Mix more (and heat more if necessary) until clear. | * Mix and then microwave this until it boils. Mix more (and heat more if necessary) until clear. | ||
* Leave to cool, then add Ethidium Bromide stock solution (small brown bottle in second drawer of cabinet) at a concentration of 2ul/40ml (so about 2.25ul for this gel) when gel is at about 50°C. | * Leave to cool, then add Ethidium Bromide stock solution (small brown bottle in second drawer of cabinet) at a concentration of 2ul/40ml (so about 2.25ul for this gel) when gel is at about 50°C. ''Ethidium bromide is a carcinogen, so wear gloves when handling.'' | ||
* Take the inner gel box, tape the ends (push down hard to seal gaps between tape and plastic) to prevent leaks, place comb in, then pour in gel before it sets (setting temp is between 30C and 40C) | * Take the inner gel box, tape the ends (push down hard to seal gaps between tape and plastic) to prevent leaks, place comb in, then pour in gel before it sets (setting temp is between 30C and 40C) | ||
*Once gel is set, remove tape and place in outer box. | *Once gel is set, remove tape and place in outer box. | ||
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* Attach electrodes to gel box, negative (black) by wells, positive (red) far end from wells, and turn on current. | * Attach electrodes to gel box, negative (black) by wells, positive (red) far end from wells, and turn on current. | ||
* After 30-45 mins, shine UV light on the gel, look through red filter to see bands. Wear googles or glasses to protect eyes from UV, and cover skin. | * After 30-45 mins, shine UV light on the gel, look through red filter to see bands. Wear googles or glasses to protect eyes from UV, and cover skin. | ||
=== Dispose of gel === | |||
* While wearing gloves, pour away buffer, and place gel in gel waste box. Thoroughly wash all gel making apparatus. |
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