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Make and run an agarose gel: Difference between revisions
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Make and run an agarose gel (view source)
Revision as of 20:06, 15 September 2012
, 15 September 2012→Make gel
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* Choose the gel concentration (higher the concentration the smaller the bands you can see). The following instructions are for a 1% of 50ml, which we normally use. | * Choose the gel concentration (higher the concentration the smaller the bands you can see). The following instructions are for a 1% of 50ml, which we normally use. | ||
* Add | * Add 0.5 gram of agarose to 5g (or 5 ml - roughly the same) of TBE 10x (you will find in big brown bottle in bottom drawer of cabinet) and 45g (or 45ml) of dH20 (we use deionized water, should be on floor or in bottom drawer of cabinet). | ||
* Mix and then microwave this until it | * 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 at a concentration of 2ul/40ml (so about 2.25ul for this gel) 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. | ||
* 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. | |||
* Make loading buffer (about 100ml for a 50ml gel), of 1xTBE (1 part TBE to 9 parts dH20. | |||
* Pour loading buffer over gel so gel is completely covered by 1-3 mm of buffer. | |||
* 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. | |||
