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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)
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=== Make gel === | === Make gel: === | ||
* 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% gel of 50ml, which we normally use. | ||
* Add 0.5 | * 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. | ||
* 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. | ||
* Make loading buffer (about 100ml for a 50ml gel), of 1xTBE | * Make loading buffer (about 100ml for a 50ml gel), of 1xTBE. This lets the current flow. | ||
* Pour loading buffer over gel so gel is completely covered by 1-3 mm of buffer. | * Pour loading buffer over gel so gel is completely covered by 1-3 mm of buffer. | ||
=== Load samples, run gel and visualise: === | |||
* Make a mix of sample and loading buffer (mix of food colouring for dye and glycerol for weight) for each sample you want to run, then pipette each mix into a well. Standard way is, if wells are at far end from you, load ladder at far left (well 1) and load samples to right of this. Quantity to load varies. Ladder quantity also varies, but for [http://www.taq-dna.com/rich_files/attachments/DNA_Ladders_DNA_Weight_markers_DNA_leiter/1000_bp_1kb_DNA_Ladder.pdf current] ladder, use 5ul ladder to 2ul loading buffer. | |||
* 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. | ||
