Berkeley LBL/KonniamNotebook

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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
  • 1uL dNTP
  • 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 (7/19/07)
Materials:

  • 1uL R-genomic DNA
  • 10uL 5x HF buffer
  • 5uL primer
  • 0.5uL Phusion
  • 25uL DMSO
  • 1uL dNTP
  • 30uL H2O

Conditions:

  • 98C 30s
  • 98C 10s*
  • 62C 30s*
  • 72C 32s 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-bchI (7/20/07) with KpnI and BglII
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)

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

6.)Transform into NovaBlue (9/3/07)
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)
8.)Miniprep cells with pET3a-R-bchH (9/14/07)
9.)Sequence