NYMU Taipei/Lab Notes/2007 9 27
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| + | ==CinR+HSL+D-term check== | ||
* After re-culture in 20-block plate,randomly pick 6 blocks from CinR+HSL+D-term plate | * After re-culture in 20-block plate,randomly pick 6 blocks from CinR+HSL+D-term plate | ||
* PCR check using VF2, VR primers | * PCR check using VF2, VR primers | ||
* PCR total 50 uL, 10 uL for GEL separation | * PCR total 50 uL, 10 uL for GEL separation | ||
| - | |||
* sample at each lane | * sample at each lane | ||
** 1Kb ladder | ** 1Kb ladder | ||
| - | ** block #1 (insert band appears) | + | ** block #1 (insert band appears), 0.41 ug/uL |
** block #2 (insert band appears, and one extra band around 400 bp) | ** block #2 (insert band appears, and one extra band around 400 bp) | ||
| - | ** block #3 (insert band appears) | + | ** block #3 (insert band appears), 0.25 ug/uL |
| - | ** block #4 (insert band appears) | + | ** block #4 (insert band appears), 0.41 ug/uL |
** 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) | ||
| - | * | + | * expected 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 | ||
* extra band at 400 bp might be plasmid failed to be correctly ligated | * 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 | ** 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]] | ||
---- | ---- | ||
| - | * [ | + | |
| + | ==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 | ||
| + | {| align=center | border=1 | style="text-align:center" | ||
| + | !glucose (%) | ||
| + | !O.D. 600 | ||
| + | !white light | ||
| + | !green light | ||
| + | !LB volume (uL) | ||
| + | !glucose volume (uL) | ||
| + | |- | ||
| + | |0 | ||
| + | |2.487 | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 0p-w.JPG|100px]] | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 0p-g2.JPG|100px]] | ||
| + | |5,000 | ||
| + | |0 | ||
| + | |- | ||
| + | |1 | ||
| + | |2.091 | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 1p-w.JPG|100px]] | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 1p-g3.JPG|100px]] | ||
| + | |4,750 | ||
| + | |250 | ||
| + | |- | ||
| + | |3 | ||
| + | |1.861 | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 3p-w.JPG|100px]] | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 3p-g.JPG|100px]] | ||
| + | |4,250 | ||
| + | |750 | ||
| + | |- | ||
| + | |5 | ||
| + | |1.637 | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 5p-w.JPG|100px]] | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 5p-g.JPG|100px]] | ||
| + | |3,750 | ||
| + | |1,250 | ||
| + | |- | ||
| + | |10 | ||
| + | |1.025 | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 10p-w.JPG|100px]] | ||
| + | |[[Image:NYMU Taipei pOmpC-EYFP 10p-g.JPG|100px]] | ||
| + | |2,500 | ||
| + | |2,500 | ||
| + | |- | ||
| + | |} | ||
Latest revision as of 12:02, 3 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), 0.41 ug/uL
- block #2 (insert band appears, and one extra band around 400 bp)
- block #3 (insert band appears), 0.25 ug/uL
- block #4 (insert band appears), 0.41 ug/uL
- block #5 (insert band does not appear, only band around 400 bp)
- block #6 (insert band does not appear, only band around 400 bp)
- expected 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 |
