IGEM:MIT/2006/Notebook/2006-8-8

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 &mu;M Salicylic Acid (40 &mu;L 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

 * 1) 6 IK A/T plates (8/7 ligation mixes 2, 3, 4, 5, 7, 8)
 * 2) 2 RBS-BAT2 3-part A/T assemblies (using 30 and 32)
 * 3) 2 THI3 topos (using old and new pcr product)
 * 4) 2 osmY-E0840 3-part A/T assemblies (using reg. cut E0840 and Gel extracted version)
 * 5) 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