Berkeley LBL/KonniamNotebook

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(Construction of pET3a-R-bchH=)
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==Construction of pET3a-R-bchHI==
==Construction of pET3a-R-bchHI==
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1.)PCR the gene bchI from Rhodobacter sphaeroides<br>
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Materials:
 +
*1uL R-genomic DNA
 +
*10uL 5x GC buffer
 +
*5uL primer
 +
*0.5uL Phusion
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*5uL DMSO
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*27.5uL H2O
 +
Conditions:
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*98C 30s
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*98C 10s*
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*63C 30s*
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*72C 1:50 min*
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*Repeat cycles with * 29x
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*72C 10 min
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*4C forever
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2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br>
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3.)Digest R-bchH (8/21/07)<br>
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*42uL PCR purified fragment
 +
*5uL NEB3
 +
*1.8uL NdeI
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*1.2uL BglII
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4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br>
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5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br>
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*4.5uL pET3a
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*12.5uL R-bchH fragment
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*2uL T4 ligase buffer
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*1uL T4 ligase
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6.)Transform into NovaBlue (8/21/07)<br>
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7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br>
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8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br>
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9.)Sequence

Revision as of 02:36, 27 October 2007

Construction of pET3A Derivatives Containing R-bchHID

Construction of pET3a-R-bchH

1.)PCR the gene bchH from Rhodobacter sphaeroides
Materials:

  • 1uL R-genomic DNA
  • 10uL 5x GC buffer
  • 5uL primer
  • 0.5uL Phusion
  • 5uL DMSO
  • 27.5uL H2O

Conditions:

  • 98C 30s
  • 98C 10s*
  • 63C 30s*
  • 72C 1:50 min*
  • Repeat cycles with * 29x
  • 72C 10 min
  • 4C forever

2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify
3.)Digest R-bchH (8/21/07)

  • 42uL PCR purified fragment
  • 5uL NEB3
  • 1.8uL NdeI
  • 1.2uL BglII

4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)

  • 4.5uL pET3a
  • 12.5uL R-bchH fragment
  • 2uL T4 ligase buffer
  • 1uL T4 ligase

6.)Transform into NovaBlue (8/21/07)
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)
8.)Miniprep cells with pET3a-R-bchH (8/23/07)
9.)Sequence

Construction of pET3a-R-bchHI

1.)PCR the gene bchI from Rhodobacter sphaeroides
Materials:

  • 1uL R-genomic DNA
  • 10uL 5x GC buffer
  • 5uL primer
  • 0.5uL Phusion
  • 5uL DMSO
  • 27.5uL H2O

Conditions:

  • 98C 30s
  • 98C 10s*
  • 63C 30s*
  • 72C 1:50 min*
  • Repeat cycles with * 29x
  • 72C 10 min
  • 4C forever

2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify
3.)Digest R-bchH (8/21/07)

  • 42uL PCR purified fragment
  • 5uL NEB3
  • 1.8uL NdeI
  • 1.2uL BglII

4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)

  • 4.5uL pET3a
  • 12.5uL R-bchH fragment
  • 2uL T4 ligase buffer
  • 1uL T4 ligase

6.)Transform into NovaBlue (8/21/07)
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)
8.)Miniprep cells with pET3a-R-bchH (8/23/07)
9.)Sequence