IGEM:MIT/2006/Notebook/2006-8-8
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General Notes
- remove plain A/T plates from 37 room (checking for plate contamination) -done
- run a gel of pchBA/etc. -done
- topo PCR THI3
- digest BAT@ with XP, 3 part assemble RBS-BAT2
- transform
- BC 18:57, 7 August 2006 (EDT): I recommend you try both with the "freeze" step (nice name!) and without it as some people don't find this to be a helpful reaction.
- received an agar stab of DH5alpha E. coli cells with the pUCP22 pseudomonas shuttle vector in it. Plated/streaked to recover cells on an Amp plate today. This is cool news
LCs
- LC all colonies in A/T LB
- LC 1-3 and 9,10 in A/T LB supplemented with correct concentration of Isoamyl Alcohol (check with Veena for concentration/volume)
- LC 4-8 and 11-15 in A/T LB supplemented with 300-350 μM Salicylic Acid (40 μL in 10 mL LC)
- FINALLY...SOME SMELL TESTS to be conducted tomorrow am!!!
Competent Indole KO Strain
- Transform with R0040.RBS.BSMT.B0015 (J45100 for B0030 and J45102 for B0032),
- Transform with R0040.RBS.ATF1-1148.B0015
- Transform with J45993.RBS.BSMT.B0015
- Transform with J45993.RBS.ATF1-1148.B0015
- Ligations from 8/7 are saved in our freezer in a tip box!!
IAGD
- Cut BAT2 with XP
- 3 part assemble with B0030:ES and A/T:EP
- 3 part assemble with B0032:ES and A/T:EP
Transformations
- 6 IK A/T plates (8/7 ligation mixes 2, 3, 4, 5, 7, 8)
- 2 RBS-BAT2 3-part A/T assemblies (using 30 and 32)
- 2 THI3 topos (using old and new pcr product)
- 2 osmY-E0840 3-part A/T assemblies (using reg. cut E0840 and Gel extracted version)
- 2 puc18 controls (using Top10 cells and IK cells)
New THI3-mut primers
- ordered primer pair 1, without the first C because it contributed to a hairpin
Primer pair 1
* Forward: 5' CATCGTAGATGCATGTACCAGTCGACAGAATTTAATC 3' Reverse: 5' GATTAAATTCTGTCGACTGGTACATGCATCTACGATG 3' * GC content: 40.54% Location: 5-41 Melting temp: 77.1°C Mismatched bases: 1 Length: 37 bp Mutation: Substitution 5' flanking region: 18 bp Forward primer MW: 11356.53 Da 3' flanking region: 18 bp Reverse primer MW: 11378.53 Da
Primer pair 2
* Forward: 5' CATCGTAGATGCATGTACCAGTCGACAGAATTTAATCG 3' Reverse: 5' CGATTAAATTCTGTCGACTGGTACATGCATCTACGATG 3' * GC content: 42.11% Location: 5-42 Melting temp: 78.0°C Mismatched bases: 1 Length: 38 bp Mutation: Substitution 5' flanking region: 18 bp Forward primer MW: 11685.74 Da 3' flanking region: 19 bp Reverse primer MW: 11667.72 Da