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

Ran 2 gels
...nothing showed up on the first gel, which was the same as the gel below, but it had RBS30 instead of 32, ATF-A instead of ATF-B, and no promoter:

Lanes:
 * 1kB ladder
 * 2-log ladder
 * RBS 32 / SAMT / term 15 in pSB1A3-1 cut with EcoRI and XbaI [from the SAMT+terminator colony A]
 * RBS 32 / SAMT / term 15 in pSB1A3-1 cut with EcoRI and XbaI [from the SAMT+terminator colony B]
 * RBS 32 / SAMT / term 15 in pSB1A3-1 cut with EcoRI and XbaI [from the SAMT+terminator colony C]
 * RBS 32 / SAMT / term 15 in pSB1A3-1 cut with EcoRI and XbaI [from the SAMT+terminator colony D]
 * ATF1-B, mutagenized, cut with EcoRI to see if the mutagenesis worked.
 * Promoter: R0040, cut with EcoRI and SpeI

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

1.5&mu;L H20

3.5&mu;LL RBS (15-25 ng/uL)

3.5&mu;LL Enzyme+B.0015 (15-25 ng/uL)

1&mu;LL T4 DNA Ligase Buffer

0.5&mu;LL T4 DNA Ligase

Transformation
The following ligation mixes were transformed:

R0040.B0030.SAMT.B0015 A, B, C, & D

R0040.B0032.SAMT.B0015 A, B, C, & D