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User:DrJones1935/29 July 2012
From 2012.igem.org
I. AGE of PCR Samples
- Make Gel:
- Measure out 0.5 g of agarose and add to a 250 mL E-flask
- Add 50 mL of 0.5x TBE buffer and swirl to mix
- Cover flask with a Kimwipe and microwave for ~1 minute until clear
- Allow to cool to ~60 oC and pour into beaker for ethidium bromide (EtBr)
- Add ~1.5 uL of EtBr stock to agarose and swirl to mix
- Immediately pour gel into tray with combs
- Allow to solidify, then remove the comb and tape and place the tray in the gel box with the wells closer to the black electrode
- Make sure it is submerged in TBE buffer
- Load Gel:
- Mix samples as drops on a piece of parafilm
- Mix:
| Ladder (Invtrogen 1 kb plus) | Blank | Sample |
|---|---|---|
| *5 uL DNA ladder *1 uL 6x Loading Dye | *8.3 uL dH2O *1.7 uL 6x Loading Dye | *6.3 uL dH2O *1.7 uL 6x Loading Dye *2.0 uL DNA |
- Load ~6 uL on gel according to the following chart:
| Lane 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|---|---|---|---|---|---|---|---|
| NEB 2-log ladder | Blank | - | + 1 | + 2 | + 3 | + 4 | + 5 |
- Store DNA at 4 oC
- Run Gel:
- Close gel box and turn on power pack
- Run gel at 30 V for 20 min to stack gel
- Run gel at 75 V until the markers have reached ~3/4 down the gel
- Image Gel:
- RESULTS:
Source: Media:Srk_2012-07-28_18hr_04min.tif
| Feature | Expected Size (bp) |
|---|---|
| Cassette | 5152 |
