NYMU Taipei/Lab Notes/2007 9 27

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(→CinR+HSL+D-term check)
 
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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
 +
* 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)
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* 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
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* References
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** [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]]
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----
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==pOmpC glucose response testing by construct (pOmpC + EYFP)==
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* design: 5 tubes for 0%, 1%, 3%, 5%, 10% extra glucose cultured in LB medium (total volume 5 mL = 5,000 uL)
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* tube with 0% extra glucose is treated as negative control
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* 20% glucose solution
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{| align=center | border=1 | style="text-align:center"
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!glucose (%)
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!O.D. 600
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!white light
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!green light
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!LB volume (uL)
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!glucose volume (uL)
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|-
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|0
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|2.487
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|[[Image:NYMU Taipei pOmpC-EYFP 0p-w.JPG|100px]]
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|[[Image:NYMU Taipei pOmpC-EYFP 0p-g2.JPG|100px]]
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|5,000
 +
|0
 +
|-
 +
|1
 +
|2.091
 +
|[[Image:NYMU Taipei pOmpC-EYFP 1p-w.JPG|100px]]
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|[[Image:NYMU Taipei pOmpC-EYFP 1p-g3.JPG|100px]]
 +
|4,750
 +
|250
 +
|-
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|3
 +
|1.861
 +
|[[Image:NYMU Taipei pOmpC-EYFP 3p-w.JPG|100px]]
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|[[Image:NYMU Taipei pOmpC-EYFP 3p-g.JPG|100px]]
 +
|4,250
 +
|750
 +
|-
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|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]

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
glucose (%) O.D. 600 white light green light LB volume (uL) glucose volume (uL)
0 2.487 NYMU Taipei pOmpC-EYFP 0p-w.JPG NYMU Taipei pOmpC-EYFP 0p-g2.JPG 5,000 0
1 2.091 NYMU Taipei pOmpC-EYFP 1p-w.JPG NYMU Taipei pOmpC-EYFP 1p-g3.JPG 4,750 250
3 1.861 NYMU Taipei pOmpC-EYFP 3p-w.JPG NYMU Taipei pOmpC-EYFP 3p-g.JPG 4,250 750
5 1.637 NYMU Taipei pOmpC-EYFP 5p-w.JPG NYMU Taipei pOmpC-EYFP 5p-g.JPG 3,750 1,250
10 1.025 NYMU Taipei pOmpC-EYFP 10p-w.JPG NYMU Taipei pOmpC-EYFP 10p-g.JPG 2,500 2,500
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