Berkeley LBL/KonniamNotebook
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==Construction of pET3A Derivatives Containing R-bchHID== | ==Construction of pET3A Derivatives Containing R-bchHID== | ||
| - | + | ==Construction of pET3a-R-bchH=== | |
| - | + | 1.)PCR bchH, bchI, bchD genes from Rhodobacter sphaeroides<br> | |
| - | + | 2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br> | |
| + | 3.)Digest R-bchH (8/21/07)<br> | ||
| + | *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)<br> | ||
| + | 5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br> | ||
| + | *4.5uL pET3a | ||
| + | *12.5uL R-bchH fragment | ||
| + | *2uL T4 ligase buffer | ||
| + | *1uL T4 ligase | ||
| + | 6.)Transform into NovaBlue (8/21/07)<br> | ||
| + | 7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br> | ||
| + | 8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br> | ||
| + | 9.)Sequence | ||
Revision as of 02:28, 27 October 2007
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
