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Contents |
Gel for the previous day's digestion
150 ml of gel prepared.
Protocol: Gel Electrophoresis
Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%.
VOL is the desired volume of gel in ml:
CH Lab
- Add 0.01*VOL g of agarose to a clean glass bottle.
- Pour VOL/50 ml of 50xTAE in a graduated cylinder. Fill up to VOL ml with di water.
- Add the resulting VOL ml of 1xTAE to the glass bottle with agarose.
- Microwave, at 7, the bottle (loose cap!) until it boils.
- Carefully remove bottle (can be super heated!) and check for the total absence of particles. Microwave again if needed.
- Prepare a gel box, with comb, and fill it up with the agarose solution (maybe not the whole solution is needed).
- Add 0.05 µl per ml of gel in the box of Red Gel (it's in the iGEM drawer) and stirr until disolved.
- Wait until cold and solidified.
- Carefully remove comb.
- Place the box in the electrophoresis chamber.
- Fill up the electrophresis chamber with 1x TAE buffer.
- Add blue dye to the DNA samples (6x loading buffer, that is 10 µl in 50 µl of DNA solution).
- Inject 30 µl of ladder marker in the first well (that's 1 µg of DNA).
- Inject 60 µl of each DNA solution in the other wells.
- Set voltage to 70-90 V and run for 30-40 min, or until the dye reaches the last 25% of the gel length (DNA travels from - to +).
- Place the gel under the camera, cover, turn UV on and take photos!
Preparing the ladder:
- get 1kb ladder DNA from the freezer (500 µg/ml).
- for 30 charges, 30 µl per charge, we need 900 µl:
- 60 µl of 1kb ladder DNA
- 150 µl of dye (6x loading buffer)
- 690 µl of water
BM Lab
In this lab the gels are slightly different. The total volumes for the small, the medium and the large gel are respectively 60ml, 80ml and 90ml. As we use 0.5x TAE buffer instead of 1x, we can use higher voltages (170V seems to work fine). The gel should run 20-40 minutes, not more. As the gel is thinner, load less DNA (up to ~10ul).
| Ladder | pMP1 | pMP2 | pMP3 | PHY1 | PHY2 | PHY3 | PHY4 | pGL1 lig1 | pGL2 lig1 | pGL3 lig2 |
|---|
- Comments
None of the bands correspond with what they should have been:
- pMP: 3655 and 1026 bp
- pGL with FseI: 4263 and 1834 bp
- pGL with MfeI: 4108 and 1989 bp
- PHY42: 5413 bp and the length of melanopsin
Purification of the digested plasmids
Done but, since the digestion happened to be screwed up, didn't bring anything. The Nanodrop showed very low concentrations and weird rations for the "cleaned" DNA.
Again digestion, just for control of DNA and enzymes
The gel had shown that something went very wrong. We decided to redo it, but using a total of 50 µl solution per digestion.
We used 5 µl of DNA and 1 µl of enzyme (except 2 µl of FseI) for each, ignoring concentrations.
General recipe:
- 5 µl of DNA
- 1 µl of enzyme
- 0.5 µl of BSA 100x
- 5 µl of buffer 10x
- Water, 37.5 µl for double digest, 38.5 for single digest
- pMP
- D1: HindIII + XbaI
- D2: HindIII
- D3: XbaI
Using Buffer N4
- pGLI
- D4: HindIII + FseI
- D5: HindIII
- D6: FseI
With buffer N2
- PHY42
- D7: NheI + XmaI
- D8: NheI
- D9: XmaI
With buffer N4
- pGLII
- D10: HindIII + MfeI
- D11: HindIII
- D12: MfeI
With buffer N4
All let 1:30 hours at 37ºC, shaking (600 rpm).
After running the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye), we concluded that both the DNA and the enzymes are fine. This means we can continue the cloning!
Gel scheme:
| Ladder | pMP1 | D1 | D2 | D3 | purified pMP1 (nothing) | pGL1 | D4 | D5 | D6 | pure pGLI |
|---|---|---|---|---|---|---|---|---|---|---|
| Ladder | D10 | D11 | D12 | pGLII pure | PHY42 | D7 | D8 | D9 |
