NYMU Taipei/Lab Notes/2007 9 27
From 2007.igem.org
(Difference between revisions)
(→pOmpC glucose response testing by construct (pOmpC + EYFP)) |
(→CinR+HSL+D-term check) |
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** block #5 (insert band does not appear, only band around 400 bp) | ** block #5 (insert band does not appear, only band around 400 bp) | ||
** block #6 (insert band does not appear, only band around 400 bp) | ** block #6 (insert band does not appear, only band around 400 bp) | ||
| - | * expect results: one band at 1,623 bp is our insert | + | * expect results: one band at (1,623 + 316) bp is our insert |
** insert = RBS+CinR (2,843 - 2,053) + RBS+HSL (2,783 - 2,053) + D-term (3,266 - 3,163) | ** insert = RBS+CinR (2,843 - 2,053) + RBS+HSL (2,783 - 2,053) + D-term (3,266 - 3,163) | ||
** insert = 790 + 730 + 103 = 1,623 | ** insert = 790 + 730 + 103 = 1,623 | ||
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[[Image:NYMU Taipei 20070927 CinR-HSL-D-term plate culture test.jpg]] | [[Image:NYMU Taipei 20070927 CinR-HSL-D-term plate culture test.jpg]] | ||
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| + | |||
==pOmpC glucose response testing by construct (pOmpC + EYFP)== | ==pOmpC glucose response testing by construct (pOmpC + EYFP)== | ||
* design: 5 tubes for 0%, 1%, 3%, 5%, 10% extra glucose cultured in LB medium (total volume 5 mL = 5,000 uL) | * design: 5 tubes for 0%, 1%, 3%, 5%, 10% extra glucose cultured in LB medium (total volume 5 mL = 5,000 uL) | ||
Revision as of 14:16, 2 October 2007
CinR+HSL+D-term check
- After re-culture in 20-block plate,randomly pick 6 blocks from CinR+HSL+D-term plate
- PCR check using VF2, VR primers
- PCR total 50 uL, 10 uL for GEL separation
- sample at each lane
- 1Kb ladder
- block #1 (insert band appears)
- block #2 (insert band appears, and one extra band around 400 bp)
- block #3 (insert band appears)
- block #4 (insert band appears)
- block #5 (insert band does not appear, only band around 400 bp)
- block #6 (insert band does not appear, only band around 400 bp)
- expect results: one band at (1,623 + 316) bp is our insert
- insert = RBS+CinR (2,843 - 2,053) + RBS+HSL (2,783 - 2,053) + D-term (3,266 - 3,163)
- insert = 790 + 730 + 103 = 1,623
- extra band at 400 bp might be plasmid failed to be correctly ligated
- PCR product length without any insert = (3,189 - 3,049) + 176 = 316
- References
- [http://partsregistry.org/Image:PsB1AK3_map.jpg vector map of pSB1AK3 and VF2 VR primers]
pOmpC glucose response testing by construct (pOmpC + EYFP)
- design: 5 tubes for 0%, 1%, 3%, 5%, 10% extra glucose cultured in LB medium (total volume 5 mL = 5,000 uL)
- tube with 0% extra glucose is treated as negative control
- 20% glucose solution
| glucose (%) | O.D. 600 | white light | green light | LB volume (uL) | glucose volume (uL) |
|---|---|---|---|---|---|
| 0 | 2.487 | 
| 
| 5,000 | 0 |
| 1 | 2.091 | 
| 
| 4,750 | 250 |
| 3 | 1.861 | 
| 
| 4,250 | 750 |
| 5 | 1.637 | 
| 
| 3,750 | 1,250 |
| 10 | 1.025 | 
| 
| 2,500 | 2,500 |
