Berkeley LBL/KonniamNotebook
From 2007.igem.org
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
