From 2012.igem.org
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I. Check Old Plates for Growth
- RESULTS: There was XL1-Blue colony growth on Kan50 plates from 15 Aug 2012 that was not present on controls for the 1:1 (~4 colonies) and 1:2 (~1 colony) vector-cassette ligations.
II. Colony PCR
- Mix according to the following table
| | 1 Reaction | Master Mix (x6)
|
| Reagent | (uL) | (uL)
|
| water | 17.25 | 103.5
|
| 5x KAPA HiFi buffer | 5 | 30
|
| 10 mM dNTP mix | 0.75 | 4.5
|
| 10 uM primer (F) | 0.75 (pBf) | 4.5 (pBf)
|
| 10 uM primer (R) | 0.75 (pBr) | 4.5 (pBr)
|
| Template | - | -
|
| KAPA HiFi polymerase | 0.5 | 3
|
- Aliquot 50 uL of mix to 6 PCR tubes
- Note: Last tube (control) had less than others
- For templates, use a pipette tip to touch a colony from one of the plates, dip it into a PCR tube, and then drop it into a culture tube as described in the next section (III). Do not use template for control.
| Step | Temp (oC) | Time
|
| 1 | 95 | 5 m
|
| 2 | 98 | 20 s
|
| 3 | 63 | 15 s
|
| 4 | 72 | 5 m
|
| 5 | GOTO 2 | 34x
|
| 6 | 72 | 5 m
|
| 7 | 4 | ∞
|
III. Start Liquid Cultures for PCR Colonies
- Make LB-Kanamycin (50 ug/mL) media
- 10 mL of LB media
- 10 uL of Kanamycin stock (50 mg/mL)
- Aliquot ~2 mL of LB-Kanamycin media to 5 culture tubes
- Drop the pipette tip from each colony PCR into a tube labeled in the same way as the PCR tubes
- Incubate overnight with shaking at ~32 oC
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