Team:UCSF/Tiffany Saw Notebook
TIFFANY SAW SUMMER EXPERIENCE
Pre-iGEM: May 12 - 15, 2008 (Week I)
May 12, 2008: Met with Andrew; set up 21 PCR rxns
May 13, 2008: Set up 5 gels, 1% agarose; 7 bands appeared; Changed conditions. 2x’s template DNA; one more band
May 14, 2008: Prepare samples for PCR in triplicate; PCR’d samples
May 15, 2008: Gels (ran twice – first ran too long)
May 19 – 22, 2008 (Week II)
May 21, 2008: Ran gel to see if old samples amplified – ¾ did (Sap 30 & PNC1, twice)
May 22, 2008: Prepared samples; Rpd3 was lost because too much dye was added; all bands present except Rpd3 (will column purify)
May 26 – 29, 2008 (Week III)
May 27, 2008: Calculated insert:vector ratio; set up charts for ligations
May 28, 2008: Set up 4 ligations be; Rpd3 could not be saved.
June 02 – 06, 2008 (Week IV)
June 02, 2008: Transformed plasmids into E. coli cells
June 03, 2008: Count colonies; Pick 3 colonies; Master plated, left toothpicks in tubes.
June 05, 2008: Digest
June 06, 2008: Ran gel
Objective: Disrupt silencing to activate a gene. Remodel chromatin so that heterochromatin can be uncoiled and opened into euchromatin so they can be activated and freely available. We’re turning on the already silenced genes to produce the desired response (shift the activated genes towards silencing) by opening heterochromatin and acetylating them to become euchromatin and open.
Rationale: In some cases, we will want differentiation to lead to gene expression (instead of gene silencing).
1. Create multiple fusions of DNA binding domains and activations domains.
2. (shared milestone with silencing group) Check if any of the combinations can sucessfully unsilence a silenced gene.
June 16 – 20, 2008 (Week 1)
June 16, 2008: Arrived at UCSF. Reviewed heteochromatin & etc.
June 17, 2008: Transformed yeast. Memory discussion.
June 18, 2008: Sent sequencing. Nanodropped samples. Memory discussions Tranformed mCherry Topo, EM3 LexA-mCherry NLS, pGal (305 vector)
June 19, 2008: Innoculated/miniprepped cultures. Received Sequencing results: all Hst4 clones are viable. Nanodropped AdhP, CycP, Sas2, LexA, VP64, mCherry, 305, Em3, PGG202LexA, EsaAdh.
June 20, 2008: PCR LexA1-261-mCherry. Jimmy & Alex’s “Rock your socks off” expo talk, plated Hst4 Cyc1& Hst4 Adh1 onto SRAF+Gal and SRAF-Gal plates.
June 23 – 27, 2008 (Week 2) June 23, 2008: Set up FACs for Lincoln samples, ran gel of LexAPEG1, LexA (SL), VP64, CM3 LexA-mCherry. Gel purified fragments of earlier gel.
June 24, 2008: Diluted FACs culture, sent in original FACs to mcachine anyways. Topo cloning.
June 25, 2008: Andrej’s talk. Feedback loops talk. EcoR1 digest of plasmid (LexA1-261-mCherry). PCR rxn of plasmid also redone, gel of samples.
June 26, 2008: Re-did digest & gel, inoculated pGal 305 cultures, transformed cultures
June 27, 2008:Prepared cultures (FRE Leu, FRE Ura), mini-prepped pGal 305 samples, double digest of LexA1-261-mCherry, nanodropped samples.
June 30 – July 04, 2008 (Week 3)
June 30, 2008: New PCR rxns of LexA1-261-mCherry with gradient heating, gel purified samples, nanodropped – results were dismal.
July 01, 2008: Mini-prepped pGal 305 and pFig samples, nanodropped, double digest of pGal and pFig, ran gel, gel purified. Innoculated cultures for microscope.
July 02, 2008: Nanodropped samples. Microscope was broken. Double digested pRS303, ran multiple ligations of pGal/pRS303/pFig. Ran gel of pGal, pRS303, and Adh1 samples, gel purification, nanodropped, transformed cells.
July 03, 2008: Plates were dismal (cultures were picked anyways, to see), mini-prepped samples, nanodrop, double digested all samples of pRS303, pGal 305, pFig, pADH1; gel, gel purify, nanodrop.
July 04, 2008: Holiday…
July 07 – July 11, 2008 (Week 4)
July 07, 2008: PCR’d LexA1-261-mCherry, gel, still looks like primer dimer.
July 08, 2008: Ran gel of other pFigs acquired form Jacob, PCR’d up LexA1-261-mCherry again and also Esa1&VP64 (now VP16), gel, gel purification, nanodropped, topo cloned Esa1 and VP16.
July 09, 2008: Esa1 & VP16 plates were generally successful, test digested Esa1 & VP16 clones, send out samples for sequencing (Hst4, pRS303 w/ pFig), mini-prepped Esa1/VP16, EcoR1 digested, gel (no DNA). Transformed successful samples. Ran gel of original pFig – too small.
July 10, 2008: Mini-prepped Jacob’s cultures, nanodropped samples, gel of vectors cut earlier, cut pFig, pGal, pCyc1, pRS303 with diff. enzymes (PSPOMI, XhoI), cut 2.5 ug of DNA, gel purified vectors of all except pFig (promoter), nanodrop.
July 11, 2008: Sequencing results came – all Esa1 sequences are viable except VP16 samples are all mutated. PCR’d up LexA1-261-mCherry with success – DMSO was added.
July 14 – July 18, 2008 (Week 5)
July 14, 2008: Nanodropped previous samples, gel purified more vectors/promoters, ligated AAR1 digested samples, ligated pCyc, pGal, and pRS303 to pFig.
July 15, 2008: Plates looked successful overall, colony PCR, gel, nanodropped, inoculated successful cultures from master plate. Set up FACs for Hst4. Made TE buffer. Ligated pFig to vectors (again).
July 16, 2008: Transformed pFig ligations into TG1 cells, miniprepped colony PCR samples, nanodropped, double digested samples (and pADH1). Double digested ligations, pCyc, and pFig. Gels of both showed that colony PCR samples were unsuccessful, pCyc-pFig ligations were correct. Mini-prepped pGal samples, inoculated 6 culture tubes of mCherry samples and VP16 samples. July 17, 2008: Mini-prepped mCherry & VP16, AARI digested samples. Ligate unsuccessful pFig ligations in the meantime, transform.
July 18, 2008: Not quite successful. Mini-prepped pRS315, LexA1-261, pFig, nanodropped those and AAR1 samples from last night. AAR1 digested mini-prepped samples, yeast transformations.
July 21 – July 25, 2008 (Week 6)
July 21, 2008: Innoculated cultures of mini-preps, but some didn’t grow – possibly too diluted? Re-did ligations and transformations of both AAR1 and pFig.
July 22, 2008: No growth on plates, too little DNA. Mini-prepped new pFig/pADH samples, also prepared LexAFL samples. Wasn’t feeling well, went home early.
July 23, 2008: Nanodropped samples from yesterday afternoon, more AAR1 digests.
July 24, 2008: Nanodropped gel-purifications form last night, double digested pRS303/pFig. Mini-prepped LexAFL, pADH1, pFig – AAR1 digested. Ligated & transformed pRS303/pFig.
July 25, 2008: Colonies on both control and ligation plates were equal – ligation repeated with CIP treatment. Mini-prepped and AAR1 digested mCherry, VP16, LexAFL, Adh1, pFig; gel purified all. AAR1 digested pFig – pFig had 3 bands… Replica plated AAR1 ligations onto SRaf + 2% Gal, SD-Leu, and SD Comp + 0.5% plates.
July 28 – Aug 01, 2008 (Week 7)
July 28, 2008: Double digest of pFig/pRS303, gel purified & CIP’d vector. Dilute FACs block (Xili help me set it up yesterday). Ligated pFig/pRS303, transformed.
July 29, 2008: Plates were 100% successful. AAR1 digested pFig once more, restreaked plates for Leeza, FloJo analysis of ligations (may have anti-silenced Cyc). Gel purified pFigs, mini-prepped pRS303/pFig ligations. Help Xili with the oscillator: transform GFP_pest, mCherry.
July 30, 2008: Innoculate cultures (for the oscillator), mini-prepped. Nanodropped last night’s mini-preps & gel purifications, test digested pRS303/pFig ligations using Alex’s surface tension gel – looks like the plasmid didn’t cut (Xili suspects the insert may be incorrect); digested samples oncemore, gel purified. Ligated/transformed oscillator samples.
July 31, 2008: nanodropped mini-preps of ligations of GFP_pest/306 (or 305) & mCherry/306 (or 305). Ligated pRS303/pFig, transformed. Test digest oscillator samples, gel (degraded on Jimmy’s gel – old gel?).
Aug 01, 2008:pRS303/pFig plates look good. Innoculated/mini-prepped. Check ADHT-305/306 using FseI/PacI (FseI appears to cut, PacI doesn’t), gel, digest O8O7_T & TetR BC, gel purify, nanodrop.
Aug 04 – Aug 08, 2008 (Week 8)
Aug 04, 2008: Double digest of Adh1T using NotI/SacI (trouble shooting for the oscillator), was misinformed (no NotI sequence in Adh1T), sending my ligations to Quintara for sequencing. Received VP64 from Geneart. Transformed VP64.
Aug 05, 2008: Miniprepping pRS303/pFig liations, test digested ligations. Ligations good, passed onto Leeza. Samples too Dilute for sequencing, sent out more samples. Innoculated VP64 samples. Aug 06, 2008: Re-cut oscillator samples with different enzymes (AscI/SpeI), still appeared off. May redesign oscillator. Mini-prepped VP64.
Aug 07, 2008: Nanodropped VP 64 mini-preps. AAR1 digested VP64. Received sequences for ADH1T (appeared normal). Trying FseI/PacI again. VP64 appeared a little big on the gel, purifying anyways. Anti-silencing/oscillator meeting. PCR’d up LexA1-261-Esa1, gel appeared dismal: template/primers were near invisible.
Aug 08, 2008: AAR1 digested pFig (305/315), PCR’d up LexA1-261-Esa1 using longer extension time, gel – no effect produced. PacI test proved to NOT linearize sample (VP64 was control)
Aug 11 – Aug 15, 2008 (Week 9)
Aug 11, 2008: Ran PCR with gradient heating, DMSO, and fresh DNTPs, nanodropped template DNA, still no results. FseI/PacI DD of ADHT, CIP ADHT, ligate ADHT (305/306). to mCherry/GFP_pest, transformed. Nanodropped AAR1 digested samples from last night. Innoculated cultures from Thursday of Leeza’s ADH1 don’t seem to grow, picking new colonies.
Aug 12, 2008: Only one tube grew, mini-prepped sample, nanodrop result was low. CIP treated FseI/PacI O/N cultures, retransformed ADH1 (from master box) into 305 (double digested with PSPOMI/XhoI). Had an AgeI assay of FseI and PacI (stopped every 1, 2, 3, 5 hours), gel
Aug 13, 2008: PCR’d LexA1-261/Esa1 using Andrej’s primer and protocol. Purifying pRS303. Mini-prepped new ADHT 305/306, double digest, ligated to mCherry & YEGFP_pest using Xili’s ligation kit, transformed PCR mystery solved: Leeza gave me LexA1-261-mCherry-Esa1 instead of LexA1-261-Esa1; redid PCR with Andrej’s conditions.
Aug 14, 2008: Picked colonies from plates, innnoculted cultures, mini-prepped samples, ran gel of samples – odd; ligating again. Mini-prepped new ADH1 samples. Double digest of ADH1, gel (odd). PCR’d sample, gel showed dismal results.
Aug 15, 2008: Double digest of oscillator samples using SacII/SpeI, gel showed samples appeared to have cut well. mCherry samples cut fine, but YEGFP appeared to be small – sending out for sequencing for final results.
Aug 17 – on
-> Sequencing was a mess for all samples, halted oscillator efforts for anti-silencing efforts.
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