NYMU Taipei/Lab Notes/2007 9 27
From 2007.igem.org
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==CinR+HSL+D-term check== | ==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 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] | |
[[Image:NYMU Taipei 20070927 CinR-HSL-D-term plate culture test.jpg]] | [[Image:NYMU Taipei 20070927 CinR-HSL-D-term plate culture test.jpg]] | ||
---- | ---- | ||
| - | + | ==pOmpC glucose response testing by construct (pOmpC + EYFP)== | |
| - | + | * design: 5 tubes for 0%, 1%, 3%, 5%, 10% extra glucose cultured in LB medium | |
| - | + | * tube with 0% extra glucose is treated as negative control | |
{| align=center | border=1 | {| align=center | border=1 | ||
!glucose (%) | !glucose (%) | ||
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|1 | |1 | ||
|2.091 | |2.091 | ||
| - | |[[Image:NYMU Taipei pOmpC-EYFP 1p-w.JPG]] | + | |[[Image:NYMU Taipei pOmpC-EYFP 1p-w.JPG|300px]] |
| | | | ||
|- | |- | ||
Revision as of 10:26, 28 September 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 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
- tube with 0% extra glucose is treated as negative control
| glucose (%) | O.D. 600 | white light | green light |
|---|---|---|---|
| 0 | 2.487 | 
| 
|
| 1 | 2.091 | 
| |
| 3 | 1.861 | ||
| 5 | 1.637 | ||
| 10 | 1.025 |
