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User:DrJones1935/5 August 2012
From 2012.igem.org
I. AGE of PCR Samples
- Make Gel:
- Measure out 0.50325 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 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 ~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 | Blank | - | 2.1 | 2.2 | 2.3 | 2.4 | 2.5 |
- 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 of gel
- Image Gel:
- RESULTS:
Source: Media:Srk_2012-08-05_13hr_47min.tif
| Feature | Expected Size (bp) |
|---|---|
| LacZ | 3151 |
II. PCR for LacZ
- Mix Reagents:
- Mix according to the following table
| 1 Reaction | Phusion Master Mix (x2) | KAPA HiFi Master Mix (x2) | KAPA LongRange Master Mix (x2) | |
|---|---|---|---|---|
| Reagent | (uL) | (uL) | (uL) | (uL) |
| water | 33 | 66 | 66 | 59 |
| 5x Appropriate Buffer | 10 | 20 | 20 | 20 |
| 10 mM dNTP mix | 1.5 | 3 | 3 | 3 |
| Mg2+ | - | - | - | 7 |
| 10 uM primer (F) | 2.5 (p2f_2) | 5 (p2f_2) | 5 (p2f_2) | 5 (p2f_2) |
| 10 uM primer (R) | 2.5 (p2r_2) | 5 (p2r_2) | 5 (p2r_2) | 5 (p2r_2) |
| Template Mix | colony | - | - | - |
| Appropriate polymerase | 0.5 | 1 | 1 | 1 |
- Touch a sterile toothpick to one colony each for samples and smear it on the bottom of a tube.
- Transfer 50 uL of each Master Mix to 6 total tubes
- Create a separate blank under Phusion conditions
- Run PCR O/N
| Step | Temp (oC) | Time |
|---|---|---|
| 1 | 94 | 4 m |
| 2 | 94 | 30 s |
| 3 | 53 | 30 s |
| 4 | 72 | 3 m 10 s |
| 5 | GOTO 2 | 7x |
| 6 | 94 | 30 s |
| 7 | 66 | 30 s |
| 8 | 72 | 3 m 10 s |
| 9 | GOTO 6 | 30x |
| 10 | 72 | 5 m |
| 11 | 4 | ∞ |
