Make and run an agarose gel: Difference between revisions
From London Hackspace Wiki
Mycoplasma (talk | contribs) |
mNo edit summary |
||
| (12 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
Good full [http://www.methodbook.net/dna/agarogel.html explanation] of the gel process. Some points from this below: | |||
* 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. | *Typically, a band is easily visible if it contains about 20 ng of DNA. | ||
* Add 1 | |||
* Mix and then microwave this until it | === Make gel: === | ||
* 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. | |||
* 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 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. | |||
* 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) | |||
*Once gel is set, remove tape and place in outer box. | |||
* Make buffer (about 100ml for a 50ml gel), of 1xTBE. This lets the current flow. | |||
* Pour buffer over gel so gel is completely covered by 2-5 mm of buffer. | |||
=== Load samples, run gel and visualise: === | |||
* 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. To check if it's working, look for bubbles coming off electrodes. 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. | |||
=== Dispose of gel: === | |||
* While wearing gloves, pour away buffer, and place gel in gel waste box. Thoroughly wash all gel making apparatus. | |||
[[Category:Biohacking]] | |||
[[Category:Guides]] | |||
Latest revision as of 21:58, 28 May 2013
Good full 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:
- 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 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.
- 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)
- Once gel is set, remove tape and place in outer box.
- Make buffer (about 100ml for a 50ml gel), of 1xTBE. This lets the current flow.
- Pour buffer over gel so gel is completely covered by 2-5 mm of buffer.
Load samples, run gel and visualise:
- 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 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. To check if it's working, look for bubbles coming off electrodes. 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.
Dispose of gel:
- While wearing gloves, pour away buffer, and place gel in gel waste box. Thoroughly wash all gel making apparatus.
