IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/29: Difference between revisions

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(Autocreate 2008/07/29 Entry for IGEM:Cambridge/2008/Notebook/Magnetic_Bacteria)
 
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==Entry title==
==Entry title==


* I0500 promoter and pSB3K3 plasmid PCR-ed following previous protocol
* 0.8% agarose gel with SyBr Green used


** Lane 4 - Hyperladder I
** Lane 5 - I0500
** Lane 6 - I0500 cut with SpeI
** Lane 8 - pSB3K3
** Lane 9 - pSB3K3 cut with EcoRI and PstI
Result:
Unknown band shown on lane 8 with a much smaller size (400bp) than expected.
Faint band on lane 6 (1200bp) corresponding to I0500 gene size.
Other lanes didn't work.
Change of Plan after Discussion with Nice Helpful Postgrads...!:
* Directly ligate gene (mms6/mamC/magA) onto plasmid pSB2K3 carrying promoter I0500 and terminators
* Incubate 8μL TOP10 competent cells in 10ml LB at 37°C for 3 hours
* Transform TOP10 with I0500 plasmid and MagA plasmid
* I0500: 1μL of biobrick DNA + 50μL TOP10
* MagA: 2μL of DNA + 50μL of TOP10
* Followed standard transformation protocol for TOP10
===PCR MagA===
* PCR MagA gene with MagA primers received from sigma
* Notice different annealing temperature for the forward and reverse primers
** 85.4°C for MagAF and 79.4°C for MagAR
* 100μM stock of primers prepared from dry primers
** Add 210μL SDW for MagAF and 417μL SDW for MagAR
* 10μM stock prepared by 10μL of 100μM in 100μL SDW




Revision as of 03:33, 4 August 2008

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Entry title

  • I0500 promoter and pSB3K3 plasmid PCR-ed following previous protocol
  • 0.8% agarose gel with SyBr Green used
    • Lane 4 - Hyperladder I
    • Lane 5 - I0500
    • Lane 6 - I0500 cut with SpeI
    • Lane 8 - pSB3K3
    • Lane 9 - pSB3K3 cut with EcoRI and PstI

Result: Unknown band shown on lane 8 with a much smaller size (400bp) than expected. Faint band on lane 6 (1200bp) corresponding to I0500 gene size. Other lanes didn't work.

Change of Plan after Discussion with Nice Helpful Postgrads...!:

  • Directly ligate gene (mms6/mamC/magA) onto plasmid pSB2K3 carrying promoter I0500 and terminators
  • Incubate 8μL TOP10 competent cells in 10ml LB at 37°C for 3 hours
  • Transform TOP10 with I0500 plasmid and MagA plasmid
  • I0500: 1μL of biobrick DNA + 50μL TOP10
  • MagA: 2μL of DNA + 50μL of TOP10
  • Followed standard transformation protocol for TOP10

PCR MagA

  • PCR MagA gene with MagA primers received from sigma
  • Notice different annealing temperature for the forward and reverse primers
    • 85.4°C for MagAF and 79.4°C for MagAR
  • 100μM stock of primers prepared from dry primers
    • Add 210μL SDW for MagAF and 417μL SDW for MagAR
  • 10μM stock prepared by 10μL of 100μM in 100μL SDW



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