IGEM:MIT/2006/Notebook/2006-7-19

Smell Cultures

1. 4 BSMT (2 SA, 2 BA) and 2 BAMT (ctrl not induced)

Sequencing Results

Positive for SAMT.B0015 C! The one we used!

Miniprep & Subsequent Steps Taken

We miniprepped each liquid culture from last night. We found out that SAMTA.B0015 was incorrect and decided to discard B0030.SAMTA.B0015 and B0032.SAMTA.B0015. We also diluted 20 uL of the indole knockout strains containing BAMT and BSMT into 10 mL of liquid culture and 40 uL of 1 M Benzoic Acid (it actually probably should have been 20 uL). We then sequenced each of the remaining minipreps and digested each one. Note that we decided to digest ATF1 mutagenesis colonies A and B with EcoRI, the site that should have been mutated in site-directed mutagenesis.

Digests

R0040

27.5 uL H20

5 uL Buffer 2

.5 uL BSA

16 uL 50 ng/uL DNA

.5 uL EcoRI

.5 uL SpeI

B0030.SAMT.B0015

27.5 uL H20

5 uL Buffer 2

.5 uL BSA

16 uL 50 ng/uL DNA

.5 uL EcoRI

.5 uL XbaI

B0030.SAMT.B0015 A, B, & C + B0032.SAMT.B0015 A, B, C, & D

35.5 uL H20

5 uL Buffer 2

.5 uL BSA

8 uL 100 ng/uL DNA

.5 uL EcoRI

.5 uL XbaI

ATF1 Mutagenesis A & B

36 uL H20

5 uL Buffer 2

.5 uL BSA

8 uL 100 ng/uL DNA

.5 uL EcoRI

Ligation

What we need to do is ligate R0040 to the SAMT.B0015 constructs that we have.

1.5μL H20

3.5μLL RBS (15-25 ng/uL)

3.5μLL Enzyme+B.0015 (15-25 ng/uL)

1μLL T4 DNA Ligase Buffer

0.5μLL T4 DNA Ligase