Berkeley LBL/KonniamNotebook

From 2007.igem.org

(Difference between revisions)
(Construction of pET3a-R-bchHI)
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==Construction of pET3a-R-bchHI==
==Construction of pET3a-R-bchHI==
-
1.)PCR the gene bchI from Rhodobacter sphaeroides<br>
+
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br>
Materials:
Materials:
*1uL R-genomic DNA
*1uL R-genomic DNA
-
*10uL 5x GC buffer
+
*10uL 5x HF buffer
*5uL primer
*5uL primer
*0.5uL Phusion
*0.5uL Phusion
-
*5uL DMSO
+
*25uL DMSO
-
*27.5uL H2O
+
*1uL dNTP
 +
*30uL H2O
Conditions:
Conditions:
*98C 30s
*98C 30s
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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-bchH (8/21/07)<br>
+
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br>
-
*42uL PCR purified fragment
+
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br>
-
*5uL NEB3
+
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br>
-
*1.8uL NdeI
+
*4.5uL pET3a-R-bchH
-
*1.2uL BglII
+
*12.5uL R-bchI fragment
-
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
*2uL T4 ligase buffer
*1uL T4 ligase
*1uL T4 ligase
-
6.)Transform into NovaBlue (8/21/07)<br>
+
6.)Transform into NovaBlue (9/3/07)<br>
-
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br>
+
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br>
-
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br>
+
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br>
9.)Sequence
9.)Sequence

Revision as of 02:41, 27 October 2007

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
  • 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
  • 25uL DMSO
  • 1uL dNTP
  • 30uL 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-bchI (7/20/07) with KpnI and BglII
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-bchH (9/14/07)
9.)Sequence