Team:Stockholm/13 September 2010

From 2010.igem.org

(Difference between revisions)
(→Cloning of N-CPPs into pSB1C3)
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Blastn alignments against [[media:Blastn_N-Tra10_pSB1C3.nCPP_11sep.txt|N-Tra10]], [[media:Blastn_N-TAT_pSB1C3.nCPP_11sep.txt|N-TAT]] and [[media:Blastn_N-LMWP_pSB1C3.nCPP_11sep.txt|N-LMWP]] indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).
Blastn alignments against [[media:Blastn_N-Tra10_pSB1C3.nCPP_11sep.txt|N-Tra10]], [[media:Blastn_N-TAT_pSB1C3.nCPP_11sep.txt|N-TAT]] and [[media:Blastn_N-LMWP_pSB1C3.nCPP_11sep.txt|N-LMWP]] indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).
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====Transformations====
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Since pSB1C3.N-TAT and pSB1C3.N-Tra10 colony samples were accidentally discarded, prepared plasmids were used to transform new cells in order to prepare glycerol stocks.
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 +
Standard transformation with 1 μl plasmid DNA.
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*pSB1C3.N-TAT
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*pSB1C3.N-Tra10
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====Sequencing====
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DNA concentrations of 11/9 plasmid preps were measured by Mimmi and samples were sent for sequencing for isolation of N-LMWP.
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*'''pSB1C3.nCCP 2''': ABS0045 B92
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*'''pSB1C3.nCCP 3''': ABS0045 B93
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*'''pSB1C3.nCCP 5''': ABS0045 B94
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*'''pSB1C3.nCCP 8''': ABS0045 B95
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*'''pSB1C3.nCCP 9''': ABS0045 B96
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*'''pSB1C3.nCCP 10''': ABS0045 B97
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*'''pSB1C3.nCCP 11''': ABS0045 B98
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*'''pSB1C3.nCCP 12''': ABS0045 B99

Revision as of 18:02, 14 September 2010

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Contents

Andreas

Preparation of Top10 chemically competent cells

None of the clones streaked onto the Amp 100 plate (11/9) grew, indicating no AmpR contamination in cells. One of the ON cultures were therefore selected for preparation of competent cells.

Procedures according to protocol. Growth conditions changed to 25 °C, 220 rpm (OD600 reached after ≈6 h).

A 100 μl aliquot was divided and spread onto Amp 100, Cm 25 and Km 50 plates to verify that they were not contaminated.

Cloning of N-CPPs into pSB1C3

Sequencing results from 8/9 returned.

  • pSB1C3.nCPP 3 (failed)
  • pSB1C3.nCPP 4 (fasta)
  • pSB1C3.nCPP 6 (fasta)
  • pSB1C3.nCPP 7 (fasta)
  • pSB1C3.nCPP 8 (fasta)

Blastn alignments against N-Tra10, N-TAT and N-LMWP indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).

Transformations

Since pSB1C3.N-TAT and pSB1C3.N-Tra10 colony samples were accidentally discarded, prepared plasmids were used to transform new cells in order to prepare glycerol stocks.

Standard transformation with 1 μl plasmid DNA.

  • pSB1C3.N-TAT
  • pSB1C3.N-Tra10

Sequencing

DNA concentrations of 11/9 plasmid preps were measured by Mimmi and samples were sent for sequencing for isolation of N-LMWP.

  • pSB1C3.nCCP 2: ABS0045 B92
  • pSB1C3.nCCP 3: ABS0045 B93
  • pSB1C3.nCCP 5: ABS0045 B94
  • pSB1C3.nCCP 8: ABS0045 B95
  • pSB1C3.nCCP 9: ABS0045 B96
  • pSB1C3.nCCP 10: ABS0045 B97
  • pSB1C3.nCCP 11: ABS0045 B98
  • pSB1C3.nCCP 12: ABS0045 B99
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