NYMU Taipei/Lab Notes/2007 9 27

From 2007.igem.org

(Difference between revisions)
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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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* '''CinR+HSL+D-term check'''
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* PCR check using VF2, VR primers
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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 total 50 uL, 10 uL for GEL separation
+
** PCR check using VF2, VR primers
 +
** PCR total 50 uL, 10 uL for GEL separation
[[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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* sample at each lane
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** sample at each lane
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** 1Kb ladder
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*** 1Kb ladder
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** block #1 (insert band appears)
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*** block #1 (insert band appears)
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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)
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** block #4 (insert band appears)
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*** block #4 (insert band appears)
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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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** expect results: one band at 1,623 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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* '''References'''
* '''References'''
** [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]

Revision as of 09:19, 27 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

NYMU Taipei 20070927 CinR-HSL-D-term plate culture test.jpg

    • 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]