Announcements Quiz Pre‐lab Lecture Review so far, colony data Genetic control elements Sequencing recap Today in Lab (Mod 2 Day 5)Announcements • No quiz next time (full day)! – Responsible for Day 5 + 6 material for Day 7 quiz • Module 1 revision due by 11 am next time • Previous quiz and FNT: ctrl + exp don’t work Æ Master Mix, cycler ctrl worked, exp. didn’t Æ primers, DNA templatepRSET-IPC pRSET-IPC*mutagenize transform in XL1-Blue DAY 2 DAY 3 transform in miniprepliquid culture BL21(DE3)mutant DNA DAY 4 DAY 4 liquid culture lyse cells and induce cells extract protein evalute proteinwith IPTG DAY 5 DAY 6 DAY 7/8Colony counts Teaching faculty plates pWhitescript: ~600 M124S (1:300): ~225 M124S (1:400): 52lac operon These three genes encode metabolic enzymes controllacI lacZ lacY lacA promoter + operator Encodes a repressor protein In turn, if lactose binds to the that binds to control area repressor, it is made inactive , turning it OFF. turning ON expression of Z, Y, A.Induction of a chosen protein controllacI lacZT7RNAP (polymerase) T7RNAP gene is expressed in presence of lactose or analogue. [IPTG]0 = [IPTG]t bla promoter is constitutively on. T7 promoter is turned on in presence of T7RNAP.genome plasmid BL21(DE3) bacterial strain DE3: bacteriophage (virus) used to integrate lac/T7RNAP into E. coli pLysS: protein that produces lysozyme, which binds to T7RNAP, reducing “leaky” expression. Retained by chloramphenicol selection.Sequencing reactions Dideoxy method: no 3’ OH Æ can’t elongate different fluorophore Run 4 rxns: (d)dT, dA, dG, dC and 3 others ddA′, A′′, C′′′, T′′′′ * = radioactive or fluorescent labelToday in Lab: Samples • Start with four DE3 samples carrying plasmid – WT – M124S – X#Z candidates 1 and 2 • After gel and sequencing analysis, pick just one X#Z to continue working with • End of day, “hand in” 6 pellets, or (3 pellets, 3 cultures, and 3 eppendorfs) to teaching facultyMIT OpenCourseWarehttp://ocw.mit.edu 20.109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use, visit:
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