From 2007.igem.org

Construction of pET3A Derivatives Containing R-bchHID

Construction of pET3a-R-bchH=

1.)PCR bchH, bchI, bchD genes from Rhodobacter sphaeroides
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