IGEM:MIT/2006/Notebook/2006-7-19: Difference between revisions
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Positive for SAMT.B0015 C! The one we used! | Positive for SAMT.B0015 C! The one we used! | ||
==Miniprep== | ==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 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. | 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== | ==Digests== |
Revision as of 11:54, 19 July 2006
Smell Cultures
- 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