NYMU Taipei/Lab Notes/2007 9 27

From 2007.igem.org

< NYMU Taipei/Lab Notes(Difference between revisions)
(→CinR+HSL+D-term check)
 
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* '''CinR+HSL+D-term check'''
+
==CinR+HSL+D-term check==
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** After re-culture in 20-block plate,randomly pick 6 blocks from CinR+HSL+D-term plate
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* After re-culture in 20-block plate,randomly pick 6 blocks from CinR+HSL+D-term plate
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** PCR check using VF2, VR primers
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* 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
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* sample at each lane
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*** 1Kb ladder
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** 1Kb ladder
-
*** block #1 (insert band appears)
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** block #1 (insert band appears), 0.41 ug/uL
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*** block #2 (insert band appears, and one extra band around 400 bp)
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** block #2 (insert band appears, and one extra band around 400 bp)
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*** block #3 (insert band appears)
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** block #3 (insert band appears), 0.25 ug/uL
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*** block #4 (insert band appears)
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** block #4 (insert band appears), 0.41 ug/uL
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*** block #5 (insert band does not appear, only band around 400 bp)
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** block #5 (insert band does not appear, only band around 400 bp)
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*** block #6 (insert band does not appear, only band around 400 bp)
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** block #6 (insert band does not appear, only band around 400 bp)
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** expect results: one band at 1,623 bp is our insert
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* expected results: one band at (1,623 + 316) bp is our insert
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*** insert = RBS+CinR (2,843 - 2,053) + RBS+HSL (2,783 - 2,053) + D-term (3,266 - 3,163)
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** insert = RBS+CinR (2,843 - 2,053) + RBS+HSL (2,783 - 2,053) + D-term (3,266 - 3,163)
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*** insert = 790 + 730 + 103 = 1,623
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** insert = 790 + 730 + 103 = 1,623
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** extra band at 400 bp might be plasmid failed to be correctly ligated
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* extra band at 400 bp might be plasmid failed to be correctly ligated
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*** PCR product length without any insert = (3,189 - 3,049) + 176 = 316
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** PCR product length without any insert = (3,189 - 3,049) + 176 = 316
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[[Image:NYMU Taipei 20070927 CinR-HSL-D-term plate culture test.jpg]]
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* References
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* '''References'''
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** [http://partsregistry.org/Image:PsB1AK3_map.jpg vector map of pSB1AK3 and VF2 VR primers]
** [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]]
----
----
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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
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==pOmpC glucose response testing by construct (pOmpC + EYFP)==
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** tube with 0% extra glucose is treated as negative control
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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)
-
{| align=center | border=1
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* tube with 0% extra glucose is treated as negative control
 +
* 20% glucose solution
 +
{| align=center | border=1 | style="text-align:center"
!glucose (%)
!glucose (%)
!O.D. 600
!O.D. 600
!white light
!white light
!green light
!green light
 +
!LB volume (uL)
 +
!glucose volume (uL)
|-
|-
|0
|0
|2.487
|2.487
-
|
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|[[Image:NYMU Taipei pOmpC-EYFP 0p-w.JPG|100px]]
-
|
+
|[[Image:NYMU Taipei pOmpC-EYFP 0p-g2.JPG|100px]]
 +
|5,000
 +
|0
|-
|-
|1
|1
|2.091
|2.091
-
|
+
|[[Image:NYMU Taipei pOmpC-EYFP 1p-w.JPG|100px]]
-
|
+
|[[Image:NYMU Taipei pOmpC-EYFP 1p-g3.JPG|100px]]
 +
|4,750
 +
|250
|-
|-
|3
|3
|1.861
|1.861
-
|
+
|[[Image:NYMU Taipei pOmpC-EYFP 3p-w.JPG|100px]]
-
|
+
|[[Image:NYMU Taipei pOmpC-EYFP 3p-g.JPG|100px]]
 +
|4,250
 +
|750
|-
|-
|5
|5
|1.637
|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
|10
|1.025
|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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