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Make and run an agarose gel: Difference between revisions
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Typically, a band is easily visible if it contains about 20 ng of DNA. | Good full [http://www.methodbook.net/dna/agarogel.html explanation] of the gel process. Some points from this below: | ||
*Typically, a band is easily visible if it contains about 20 ng of DNA. | |||
=== Make gel: === | === Make gel: === | ||
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* 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 | * Make buffer (about 100ml for a 50ml gel), of 1xTBE. This lets the current flow. | ||
* Pour | * Pour buffer over gel so gel is completely covered by 2-5 mm of buffer. | ||
=== Load samples, run gel and visualise: === | === 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. Easier to see what you're doing when loading samples with something black under the box. | * Make a mix of sample and loading buffer (0.2 volumes loading buffer per sample - 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. Easier to see what you're doing when loading samples with something black under the box. | ||
* Attach electrodes to gel box, negative (black) by wells, positive (red) far end from wells, and turn on current. Power supply runs at about 75V. | * Attach electrodes to gel box, negative (black) by wells, positive (red) far end from wells, and turn on current. Power supply runs at about 75V. | ||
* 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. To see better, put something black under the box. Use a good camera, try long exposure. | * 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. To see better, put something black under the box. Use a good camera, try long exposure. | ||
