Berkeley LBL/KonniamNotebook

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(→Construction of pET3a-R-bchHI)
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==Construction of pET3A Derivatives Containing R-bchHID==
==Construction of pET3A Derivatives Containing R-bchHID==
-
==Construction of pET3a-R-bchH==
+
===Construction of pET3a-R-bchH===
1.)PCR the gene bchH from Rhodobacter sphaeroides<br>
1.)PCR the gene bchH from Rhodobacter sphaeroides<br>
Materials:
Materials:
Line 8: Line 8:
*0.5uL Phusion
*0.5uL Phusion
*5uL DMSO
*5uL DMSO
 +
*1uL dNTP
*27.5uL H2O
*27.5uL H2O
Conditions:
Conditions:
Line 35: Line 36:
9.)Sequence
9.)Sequence
-
==Construction of pET3a-R-bchHI==
+
===Construction of pET3a-R-bchHI===
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br>
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br>
Materials:
Materials:
Line 42: Line 43:
*5uL primer
*5uL primer
*0.5uL Phusion
*0.5uL Phusion
-
*25uL DMSO
+
*2.5uL DMSO
*1uL dNTP
*1uL dNTP
*30uL H2O
*30uL H2O
Line 48: Line 49:
*98C 30s
*98C 30s
*98C 10s*
*98C 10s*
-
*63C 30s*
+
*62C 30s*
-
*72C 1:50 min*
+
*72C 32s min*
*Repeat cycles with * 29x
*Repeat cycles with * 29x
*72C 10 min
*72C 10 min
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2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br>
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br>
-
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br>
+
3.)Digest R-bchI with KpnI and BglII(7/20/07)<br>
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br>
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br>
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br>
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br>
Line 64: Line 65:
6.)Transform into NovaBlue (9/3/07)<br>
6.)Transform into NovaBlue (9/3/07)<br>
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br>
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br>
-
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br>
+
8.)Miniprep cells with pET3a-R-bchHI (9/14/07)<br>
 +
9.)Sequence
 +
 
 +
===Construction of pET3a-R-bchHID===
 +
1.)PCR the gene bchD from Rhodobacter sphaeroides (7/20/07)<br>
 +
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)<br>
 +
3.)Digest R-bchD with SpeI and NsiI (7/23/07)<br>
 +
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/23/07)<br>
 +
5.)Ligate R-bchD with pET3a-R-bchHI that is already digested with SpeI and NsiI(9/17/07)<br>
 +
*4.5uL pET3a-R-bchHI
 +
*12.5uL R-bchD fragment
 +
*2uL T4 ligase buffer
 +
*1uL T4 ligase
 +
6.)Transform into NovaBlue (9/17/07)<br>
 +
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br>
 +
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

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