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Revision as of 06:36, 27 October 2007 (view source) KonniamChan (Talk | contribs) (→Construction of pET3a-R-bchHID) ← Older edit		Latest revision as of 06:37, 27 October 2007 (view source) KonniamChan (Talk | contribs) (→Construction of pET3a-R-bchHI)
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	*5uL primer		*5uL primer
	*0.5uL Phusion		*0.5uL Phusion
-	*25uL DMSO	+	*2.5uL DMSO
	*1uL dNTP		*1uL dNTP
	*30uL H2O		*30uL H2O

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Latest revision as of 06:37, 27 October 2007

Contents 1 Construction of pET3A Derivatives Containing R-bchHID 1.1 Construction of pET3a-R-bchH 1.2 Construction of pET3a-R-bchHI 1.3 Construction of pET3a-R-bchHID

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