Berkeley LBL/KonniamNotebook
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*5uL primer | *5uL primer | ||
*0.5uL Phusion | *0.5uL Phusion | ||
| - | * | + | *2.5uL DMSO |
*1uL dNTP | *1uL dNTP | ||
*30uL H2O | *30uL H2O | ||
| Line 97: | Line 97: | ||
6.)Transform into NovaBlue (9/17/07)<br> | 6.)Transform into NovaBlue (9/17/07)<br> | ||
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br> | 7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br> | ||
| - | 8.)Miniprep cells with pET3a-R- | + | 8.)Miniprep cells with pET3a-R-bchHID (9/19/07)<br> |
9.)Sequence | 9.)Sequence | ||
Latest revision as of 06:37, 27 October 2007
Contents |
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
- 2.5uL 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 with KpnI and BglII(7/20/07)
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-bchHI (9/14/07)
9.)Sequence
Construction of pET3a-R-bchHID
1.)PCR the gene bchD from Rhodobacter sphaeroides (7/20/07)
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*
- 62C 30s*
- 72C 1 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 (7/20/07)
3.)Digest R-bchD with SpeI and NsiI (7/23/07)
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/23/07)
5.)Ligate R-bchD with pET3a-R-bchHI that is already digested with SpeI and NsiI(9/17/07)
- 4.5uL pET3a-R-bchHI
- 12.5uL R-bchD fragment
- 2uL T4 ligase buffer
- 1uL T4 ligase
6.)Transform into NovaBlue (9/17/07)
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)
8.)Miniprep cells with pET3a-R-bchHID (9/19/07)
9.)Sequence
