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User:DrJones1935/3 August 2012
From 2012.igem.org
I. AGE of PCR Samples
- Make Gel:
- Measure out 0.40136 g of agarose and add to a 250 mL E-flask
- Add 50 mL of 0.5x TBE buffer and swirl to mix (0.8% agarose gel)
- 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 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) | Sample |
|---|---|
| *5 uL DNA ladder *1 uL 6x Loading Dye | *6.3 uL dH2O *1.7 uL 6x Loading Dye *2.0 uL DNA |
- Load ~10 uL on gel according to the following chart (except ladder which is 6 uL):
| Lane 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|---|---|---|---|---|---|---|---|
| NEB 2-log ladder | - | PP 2 | PP 5 | PP 10 | GE 2 | GE 5 | GE 10 |
- 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 bottom of the gel
- Image Gel:
- RESULTS:
Source: Media:Srk_2012-08-03_14hr_12min.tif
| Feature | Expected Size (bp) |
|---|---|
| Cassette | 5152 |
II. Cross-over PCR
- Template Mix Specifications:
| Template | Size (bp) | Amount Wanted (pmol) | Amount Wanted (ng) | Concentration (ng/uL) | Volume (1x) | Volume (5x) |
|---|---|---|---|---|---|---|
| LacZ | 3151 | 0.00745 | 15.24 | 11.468 | 1.33 | 6.7 |
| CAT* | 729 | 0.00745 | 3.52 | 35.256 | 0.1 | 0.5 |
| tetR | 1265 | 0.00745 | 6.12 | 37.787 | 0.16 | 0.8 |
| total | 8 uL |
- *Calculations made with [http://molbiol.edu.ru/eng/scripts/h01_07.html this website]
- Mix Reagents:
- Mix according to the following table
| 1 Reaction | Master Mix (x6) | |
|---|---|---|
| Reagent | (uL) | (uL) |
| water | 31.4 | 188.4 |
| 5x KAPALongRange buffer | 10 | 60 |
| 10 mM dNTP mix | 1.5 | 9 |
| 10 uM primer (F) | 2.5 (pBf) | 15 (pBf) |
| 10 uM primer (R) | 2.5 (pBr) | 15 (pBr) |
| Template Mix | 1.6 | - |
| KAPA LongRange polymerase | 0.5 | 3 |
- Before adding template, move 48.4 uL of Master Mix to a clean PCR tube and add 1.6 uL water for negative control
- Add templates to Master Mix according to Template Specifications above, mix by pipetting
- Aliquot 50 uL of mix to 5 PCR tubes
- Run PCR O/N
| Step | Temp (oC) | Time |
|---|---|---|
| 1 | 94 | 4 m |
| 2 | 94 | 30 s |
| 3 | 54 | 30 s |
| 4 | 68 | 5 m |
| 5 | GOTO 2 | 7x |
| 6 | 94 | 30 s |
| 7 | 66 | 30 s |
| 8 | 68 | 5 m |
| 9 | GOTO 6 | 30x |
| 10 | 72 | 5 m |
| 11 | 4 | ∞ |
III. Streak out ECNR2 Plate
- Streak ECNR2 onto an LB plate from glycerol stock
- Place in 34 oC static incubator overnight
