Berkeley LBL/KonniamNotebook

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Revision as of 02:42, 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*
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 (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

@@ Line 48: / Line 48: @@
 *98C 30s
 *98C 10s*
-*63C 30s*
+*62C 30s*
-*72C 1:50 min*
+*72C 32s min*
 *Repeat cycles with * 29x
 *72C 10 min