Laboratory Notebook

5/23/11 – Mini-Prep Procedure Explained by – Kevin Attended by – Steph, Sana, Ethan, Akanksha, Boris Centrifuge (Angus Flexifuge) Overflows ano; Makes a mess with spin columns volume>500uL Protocol 25 -  Preparation and Transformation of Competent E. Coli using CaCl2 (pg 1.116) (Source – Molecular Cloning V1 – Seabrook and Russell) Materials – CaCl2-2H20 (1M) or standard transformation buffer, MgCl2-CaCl2 solution (ice cold) Media -   LB or SOB medium for initial growth of culture SOB agar plates containing 20uM MgSO4 and appropriate antibiotic SOC medium (appox. 1mL for each transformation) Nuc. Acids – Plasmid DNA Equipment – Polypropylene tube (50mL, chilled in ice) Polyprep tubes (17x100mm, chilled in ice) Water bath preset to 42 degrees 5/24/11 Tet+Amp Control had growth, Kan Control had no growth. BB5 No growth. Everything Else had growth. Tet media was 10x too little Tet. Recultured Tet controlled and transform into proper Tet. Miniprepped and streaked Amp control and BB1. Restriction of miniprep with:   Stock 1 Rxn 3 Rxn XbaI SpeI ddH20   4.5uL 13.5uL     1x Buffer 4 10x 2uL 6uL     1x BSA .1mg/uL 10x 2uL 6uL     1 ug DNA .1ug/uL 10uL       10 units XbaI 20000u/mL .5uL 1.5uL     10 units SpeI 10000/u/mL 1uL 3uL     Total   20uL 60uL     1 2 3 4 5 6 7 8 9 10 L L   MP2 R1 R2         5uL 10uL                 MP1 – Miniprep BB1 MP2 – Miniprep Amp Control R1 – Restricted MP1 R2 – Restricted MP2 Plated everything that came out Future – If it looks good grow up and rerun restrictions with better spaced restriction sites. 5/25/11 All plates made on 5/24/11 grew into lawns XbaI Digest 5uL H20 2uL buffer 4 2uL BSA 10uL DNA 1uL Enzyme (20 units) x2 samples Then warm room 30 minutes. The backbone for ES1 is pSBIAK8 which is 3426bp. The Brick for ES1 is 46bp, so the final plasmid should be around 3500bp. XbaI cuts at 1272 and 2288 of backbone, which should give fragments of 1000bp and 2500bp (according to NEB cutter). The gel is consistent with this if we assume MP1MP2 5/26/11 Results of seconds transformations 10am - AMP plates grew, no growth on Kan/Tet controls, no growth in Kan plate with ES5, and no growth in Tet plate ES2. Amp control has colonies (positive control with DNA) 1pm – Neil subcultured the two ccdB tubes that Kevin asked me to. They are labeled and in the roller. There is a mark on the tubes to indicate what tube was subcultured from what tube. We are taking 5 AMP plates and are going to perform streak-outs on them of the following plates – ES(1,3,4,6,7) from 5/25/11. Streak-out plates were divided into 4 quadrants and we took four colonies from each plate. Purpose – Separating individual colonies before additional miniprepping. 5/30/11 Experiment – Overnights of ES(1,3,4,6,7) on LB+AMP, put in incubator (37 degrees C) at 1208pm. Single colony used is circled in place. Colony counts from 5/25 transformation – TMTC=Too many to count ES1 0 ES2 TMTC ES3 TMTC ES4 40 ES5 0 ES6 5 ES7 2 6/1/11 Protocol – Pouring Agar Plates (from online source) Moving Plates – AMP – (1000xAMP); conc final – 100ug/mL. (100mg AMP/1000mL=100ug/mL) Tet – (sus 1mL) – 2.5/5000mL = 50 sus/mL LB Media 10g Bacto-tryptone (tryptone peptone)pancreatic digest of casein 5g Bacto-yeast extract (DFCO brand) 10g NaCl (red bp) .5mL 10N NaOH (not necessary for plates) For plates add 20g agar/litre Autoclave 40’ AMP Plates 2 Liters LB Media made 20g tryp 10g yeast extract 20g NaCl 40g Agar/Litre Tet Plates 5g Bacto-tryptone (tryptone peptone) 2.5 bacto yeast extract 5g NaCL (.5L) For plates at 10g agar/litre 6/2/11 Made glycerol stocks of ccdB resistant-KanControl (600uL culture+600uL 80% glycerol) Plated lovTAP stabs, streaked with toothpicks onto ChI plates. Made ccdB resistant competent cells. Concentration of CaCl2 is not on bottle. Diluted overnight culture at 1:20 into 15mL LB at 1130am. Made 100mm CaCl2 (stock 1M) Calculations for 80mm MgCl2 20mM CaCl2 solution (MgCl2 stock 2M, CaCl2 stock 1M) Final volume – 10mL C1V1=C2V2, 1MV1=.02M10mL, 2MVa=.08M10mL, V1=.2mL CaCl2, Va=.4mL MgCl2, 9.4mL H20 Backbone Labels – 1I-4A5-SW 5C-2K3-MV 7c-3T5-AV Transformations (4pm) ES2 (Tet) 4A5 (Amp) 2K3 (Kan) 3T5 (Tet) Amp+Control Kan+Control Amp-Control Kan-Control Tet-Control Protocol – Thaw comps on ice, aliquot 50uL per transformation, add 2uL DNA, incubate on ice for 20min Add 200uL LB, incubate at 37 degrees C for 3 hours Plate on antibiotic plates (50uL for amp, 100uL for kan/tet) Notes – 200uL LB added at 430pm, cells plated at 700pm 6/6/11 ES3,4,5 PCR – all primers diluted to 100uM and then a tube of 10uM node. For 50uL reaction volume x4 5uL 10x ThermoPol Buffer20uL 1ul 10(40mM) dNTPS4uL 1uL 10uM reverse primer4uL 1uL DNA .5uL Vent2uL 1uL 100mM MgSO44uL 39.5 ddH2O158uL DNA in kit is ~10mg resuspended in 10uL 50 1uL.001ug=1ng Digestions – ES1,3,4,6,7 with Ecoli+PstI, also 8,9,C Will use: NEB Buffer 4 5uL/rxn45uL 100ug/mL BSA 5uL/rxn45uL 1uL EcoRI-HF 1uL/rxn9uL 1uL PstI 1uL/rxn9uL 50uL Reaction volume 5uL DNA 5uL/rxn Total 17uL/rxn H20 33uL/rxn297uL Quick Purification of 5100L*1/250uLPH=2.0uL pH indicator 250uL buffer+50uL PCR Buffer PB has accidentally too much pH indicator (6x too much) – may gel new kit Digestions Used 2uL enzyme - ES6EcoRI-HF+XbaI, ES7SpeI+PstI Went in 20 minutes after first 2, used 3 uL Enzyme - PCR Purified ES3XbaI+PstI+DynI, PCR Purified ES4EcoRI-HF+SpeI+DpnI All digestions done in NEB4 with BSA as yesterday Ligations (x3) Master mix 3.5uL H2O10.5uL .5uL T4 Ligase1.5uL 1uL T4 Buffer3uL 1uL Vector 4uL Insert Ligation Transformations 2uL on 48uL of comp cells, started incubation for 20mins at 346pm PCRed at 405pm in incubator 6/8/11 Distribution kit contains extras of ES2. Took 1 and labeled it ES2-2. Found a possible alternative to ES5. Took it and labeled it ES5-2 (Brick#I12035). Also took VioABCE and VioABDE (Light and dark green respectively and labeled them ESLG and ESDG respectively. Ran another transformation with grow up phase and some parts in IPTG. ES5 – Kan+IPTG (added 20uL 100mM IPTG to each IPTG plate [20uL IPTG+80uL H2O) ES5-2 – Kan+IPTG ESDG – Kan ESLG – Kan ES2-2 – Tet -Kan Control +Kan Control -Tet Control Going to give 3hr grow up time at 37 degrees in LB (+IPTG for ES5, ES5-2, put in at 1145) 6/9/11 Miniprepped L3, L7D – KL Digested with EcoRI-HF+PstI using previous protocol 1% agarose gel – .8g agarose to 40mL of 1x TAE stock in 250mL bufferAdd 40mL 1x TAE Buffer to melted solutionAdd 5uL of 10mg/mL Ethidium Bromide 6/10/11 Remake Gel and rerun. Digest (makes for 17 rxns) NEB4 5uL/rxn85uL 100ug/mL BSA 5uL/rxn 85uL EcoRI-HF 1uL/rxn17uL PstI1uL/rxn17uL H2O 33uL/rxn561uL DNA 5uL/rxn 200pm 20 Digests+MP5Sill for 20 minutes at 80 degrees C 4 Dye   7/5 Digest L4 PCR with SyeI 50uL rxn vol (x6.6) 5uLà33uL 5uLà33uL 1uLà6.6uL 10uLà66uL 29uLà191.4uL 37­oC @1217 50oC @1400 7/5 Gel Digests coated 10uL Rest coated 4uL 7/5 Started cultures of L9 and L6 in LB Amp Redoing PCR of L4 minipreps 50uL rxn volume (x2.2) 5uL 10x Thermopolà11uL 1uL 40um dNTPà2.2uL 1uL 10um VPZà2.2uL 1uL 10um VRà2.2uL 1uL DNA .5uL ventà1.1uL 1uL 100um MgSO4à2.2uL 39.5uL ddH2Oà86.9uL Dilute L4-C and LF-D 1:10 Use straight for A/B Not using E/F L6 PCR 50uL rxn vol (x3.3) 1uL 40mm dNTPsà3.3uL 1uL 10um VF2à3.3uL 1uL 10um VRà3.3uL 1uL DNA .5uL ventà1.65uL 1uL 100mm MgSO4à3.3uL 39.5uL ddH2Oà130.35 5uL 10x Thermopolà16.5 7/6 L6 PCR Gel Gel shows PCR product between 200uL and 300bps which is bad. Started liquid cultures of 6 more L6 colonies from transformation plate. Ignoring satellite colonies 7/7 Miniprepped new L6 Cultures PCR of L4 (1-6) minipreps 50uL rxn volume (x6.6) 5uL 10x thermopolà33uL 1uL 40mm dNTPsà6.6uL 1ul 10mm VP2à 1ul 10um VRà 1ul DNAà .5ul ventà 1uL 100mM MgSO4à 39.5 ddH2Oà260.7uL Sequencing (macrogen) Primer made 2 pmol/k (?)(1:5 of PCR conc.) Add 4uL primer to 10uL miniprep in strip tube Label tubes 1-8 Tell Janet what 1-8 are (include primer) PCR of L3-3 ES1, LtrpR-B, LtrpR-E 50uL (x4.4) 5uL 10x thermpolà22 1uL 40mM dNTPsà4.4 1uL 10uM VP2à4.4 1uL 10uM VRà4.4 1uL DNA .5uL vent à2.2 1uL 100mM MgSO4à4.4 uL 39.5 ddH2Oà150uL L5 Digestions Digest L4 PCR Product with ECORI, NheI, DpnI Digest L3 PCR Product with XbaI, PstI, DpnI Digest PSBIC3 Digestion with DpnI 50uL rxn vol (2.2x) 5uL 10x NEB4à11uL 5uL 10x BSAà11uL 3uL enzyme 10uL DNA 27uL ddH2Oà59.4 For pSBIC3 system, just add 1uL DpnI to mix L5 Ligation mix 21uL rxn Volume 2uL T4 LIgase Buffer 3uL L4 PCR Digest END 3uL L3 PCR D XPD 2uL pSBIC3 PCR D EPD 10uL ddH2O 1uL T4 Ligase Transformations: L5 (pSBIC3, L3, L4, PCR Digests) EL7-2 (L3-3, L4-A, EAmp-Control, Ch1-Control) Using standard protocol 7/12 50uL rxn vol (2.2x) 5uL 10x thermopolà11.0 1uL 40mM dNTPsà2.2 1uL 10um VR2à2.2 1uL 10um VRà2.2 1uL DNA .5 uL ventà1.1 1uL 100mM MgSO4à2.2 39.5 uL ddH2Oà86.9 Digest of L7, L3 (EX) 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL XbaI 5uL DNA (A little short on L7) 33uL ddH2O PCR Purifying L7/L3 digests since “inserts” are very small Digest of L6, ES1, PCR with 50uL rxn vol 5uL 10x MB4 5uL BSA 1uL EcoRI-HD 1uL SpeI 1uL DpnI 5uL DNA 32uL ddH2O 7/12 Ligations 5,8 Ligation 8 – L6 Insert, L7 Backbone Ligation 5 – L4 Insert, L3 Backbone Ligation Mix – 20uL rxn vol 2uL 10x T4 ligase buffer 1uL T4 Ligase 1uL backbone 3uL insert 13uL ddH2O DNA Heat shock – 5min Transformations 5,8 50uL 5 and 8 Transformed L5 and L8 using standard protocol ES7 minprep using standard protocol. 7/13 B1006-T1 plate 1 well  4H B1006-T2 plate 1 well 4B (both are pSB1AK3) Transformed using standard protocol ES4 positive control Make glycerol stocks of L3-3, L4-A, L7-2, LtrpR-G, L6-2 Digestions – ES7-EX, ES7-SP, trpR-E PCR, XPD 50uL rxn vol 5uL NEB4 5uL BSA 1uL enzyme1 1uL enzyme 2 (1uL enzyme3) 10uL DNA 27uL ddH2O (1uL ddH2O) -37 degrees C ES7 Digestions PCR Purified trpR-E 80 degrees C Ligations 9 and 11 20uL rxn vol 2uL T4 ligase buffer 1uL backbone 3uL insert 1uL T4 Ligase 13uL ddH2O L9 insert ES1 DESD Backbone ES7 DEX L11 Insert trp-E PCR DXPD Backbone ES7 DSP Started liquid cultures of L5 and L8, 2 each 7/14 L5A-B, L8A-B miniprep using standard protocol Miniprepped T1+T2 using standard protocol 7/14 Phusion PCR 98 degrees C 30 sec (30 cycles of next 3) 98 degrees C 5 sec 63 degrees C 10 sec 72 degrees C 15 sec 72 degrees C 5 min 4 degrees C hold 50uL rxn vol 25uL phusion master mix 2.5uL VP2 primer 2.5uL VR Primer 1uL DNA 19uL dd H2O Digest T2 with EX 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL XbaI 5uL DNA 33uL ddH2O   7/14 T1 PCR digestion ESD 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL SpeI 1uL DpnI 10uL DNA 27uL ddH2O L9 and L11 transformation – Colony count Negative control – 0 Positive control – a lot ES7 DSP – 41 trpR DX – 0 L9 ~5 ES1 PCR DES – 22 ES7 DEX – 37 L11 – 120 7/15 Miniprepped L11A-P and L9 1-4 using standard protocol L9-2 messed up 7/18 PCR mRFP 50uL rxn vol 25uL 2x phusion master mix 2.5uL VR2 primer 2.5uL VR Primer 1uL DNA 19uL ddH2O RBS digestion n with SP (ES1)T7 with EX 50uL rxn bol 5uL NEB4 5uL 10x BSA 5uL DNA 23uL ddH2O 1uL enzyme 1 1uL enzyme 2 PCR of ES9 50uL rxn vol 25uL phusion 2x master mix 2.5uL VF2 primer 2.5uL primer 1uL DNA 19uL ddH2O PCR purification RFP and ES9 ES9 and mRFP digestion ES9 with ES RFP with XP 50uL rxn vol 5uL NEB 4 5uL 10x BSA 1uL DPnI 10uL DNA 1uL enzyme 1 1uL enzyme 2 27uL H2O trpR digestion with XP 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL PPNI 20uL DNA 1uL XbaI 1uL PstI 17uL H2O 7/18/11 Made LB/Chlorinphenacol plates and LB/Amp plates (2L each) using this procedure – 20g tryptone peptone 10g bacto-yeast  20g NaCl 2L dH2O 40g Agar Autoclave for 1 hour at 40 Phosphatone of ES7 and ES1 -take out 6mL of DNA -add 5mL of phosphatase buffer Add 1mL phosphatase 1hr @ 37 degrees C 5min@ degrees C 7/19 Ligations 18,19 20rxn vol 2uL T4 ligase buffer 1uL backbone insert 1uL T4 ligase 13uL ddH20 L18 8uL ES7 backbone 1uL mRFP insert L19 3uL ES9 insert 1uL ES1 backbone PCR L11 A-H 50rxn vol 25mL phusion master mix 2.5uL VF primer 2.5uL VR Primer 1uL DNA 19uL DNA Transformed cells using standard protocol Miniprepped ES7 using standard protocol Colony PCR 20uL rxn vol 10uL ddH2O 10uL Phusion 1 Colony Made cultures of L18 and L19 transformation Transformation/# of colonies L18 – 1 L19 – 2 +control – 8 ES7 DEX  -12 ES7 DSP – 2 -control – 0 mRFP – 0 ES9 DESD – 0 7/25 Buffer - 1g PEG 6000 .15mL 2M MgCl2 .5mL DMSO 9mL LB pH to ~6.5 filter sterilize L5 and T1 PCR digestion L5 with SP T1 with DXP 30 rxn vol 5uL NEB4 5uL 10XBSA 1uL DDNI 10uL DNA 1uL enzyme1 1uL enzyme2 27uL H2O PCR LovTap 50uL rxn vol 25uL phusion buffer 22uL ddH2O 1uL DNA 1uL primer 1 1uL primer 2 Digested with JpnI L11 done L12 resequence L17 bad because ptrpL in ES9 resequence ES7 Digestion with SP 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL DNA 1uL enzyme1 1uL enzyme 2 28uL H2O Kill @80 degrees C 20 minutes Phosphatase of L5 50uL rxn vol 5uL phosphatase buffer 1uL phosphatase 44uL ddH2O -1hr 37 degrees C -5min kill 65 degrees C Ligate L17 20uL rxn vol 2uL T4 Ligase buffer 1uL backbone 3uL Insert 1uL T4 Ligase 13uL ddH2O Transformed L17 and L11 PCR L8 A1 50uL rxn vol 25uL 2x phusion master mix 2.5uL VF2 Primer 2.5uL VR Primer 1uL DNA 19uL H2O L8A2 and ES7 digestion (L8A-2 and ES7-EX) 50uL rxn vol 5uL NEB4 5uL 10x BSA 10u DNA 1uL enzyme 1 1uL enzyme 2 28uL H2O PCR Purify ES7 and L8A1 Phosphatase ES7 – 1hr @37 degrees C L8A1 digestion DES 50uL rxn vol 5uL NEB4 5uL 10x BSA 10uL DNA 1uL enzyme 1 1uL enzyme 2 27uL ddH2O 1uL DPN1 80 degrees C Kill ES7 Phos L8A1 DDES   Ligations 3,7 (again) PCR ES3, ES4 with VR, VF2 Rxn mix (5x) 5uL 10x bufferà25uL 1uL 40mm dNTP (10mm each)à5uL .5uL 100um VF2à2.5uL .5uL 100um VRà2.5uL 1uL plasmid (once with straight, once 1:10 dilution) 1uL Ventà5uL 40uLddH2Oà200uL 1 uL MgSO4à5uL Thermocycler settings 95 degrees C 2 min (30 cycles of the rest) 95 degrees C 30 sec 50.3 degrees C 30 sec 74.0 degrees C 1 min 74.0 degrees C 5min 4 degrees C hold Digest of ES6, ES7 ES6 digested with EcoRI+XbaI ES7 digested with  PstI+SpeI 50uL rxn vol 5uL 10x NEB buffer 4 5uL 10x BSA 1uL enzyme 1 1uL enzyme 2 10uL DNA (from miniprep) 28uL ddH2O 2 hour incubator at 37 degrees C 20 min incubator at 80 degrees C Add 5uL Antarctic 10x buffer, 1uL Antarctic Phosphatase (Take 6uL aliquot for control-unphosphorylated) 1 hour incubation at 37 degrees C 20 min activationat 80 degrees C Note – grey dry bath high ~4 give 80 degrees C Ligations 3,7 PCR Purification of 40uL product as per qiagen kit. (extra 10uL will be used to check kit yield) Digest of ES3, ES4 (PCR products) ES3 digested with XbaI and PstI ES4 digested with EcoRI-HF and SpeI Digest mix 50uL rxn volume 5uL NEB4 (10x) 5uL 10x BSA 1uL enzyme 1 1uL enzyme 2 1uL DpnI 10uL DNA 27uL ddH2O Incubate at 37 degrees C for 2 hours, make another set to digest for 1hr. inactivate at 80 degrees C for 20min 1% agarose gel 40 wells made. 80mL/TAE. 8g Agarose (gel is a bit thin, next time go for 100mL) Ligations 3 and 7 Ligation 3 – ES3+7 Ligation 7 – ES4+6 Rxn Mixture – general 1x T4 Buffer DNA ddH2O T4 Ligase .5uL/10uL rxn vol 10 or 20uL rxn vol DNA  - NEB recommends 50ug vector and 1:3 vector:insert Consensus OWW is 10ug and 6:1 vector:insert OWWW says vector:insert is 1:1-6, all seem to work ok OWW recommends 10uL rxn vol NEB says 20uL OWWà30min incubation at room temp NEBà10min at same cond. Aliquotted T4 Buffer into 100uL tubes (ATP is not stable) Ligation 3 – 20uL rxn vol 2uL 10x T4 Buffer 1uL ES7D 1uL ES3 2hr Digest 1uL T4 Ligase 15uL H2O 1uL ES7 Digest 3uL ES3 13uL H2O 2uL ES7 4uL ES3 11uL H2O Ligation 7 – 20uL rxn vol 2uL 10x T4 buffer 1uLES6 1uL ES4 15uL H2O 1uL T4 Ligase 1uL ES6 3uL ES4 13uL H2O 2uL ES6 4uLES4 11uL H2O Incubate at room temp for 15min Inactivate at 65 degrees Cfor 15 min Transform using standard protocol (2uL DNA into 50uL comps with 20min incubation on ice with following controls ddH2O –control ddH2O+control ES3 digest ES4 Digest ES6 Digest ES7 Digest Plated 35uL on LB amp plates Phosphates ES6 digest ES6 Digest UPL 2uL 10x T4 Buffer 2uL DNA 15uL H2O 1uL T4 Ligase Incubate at room temp for 10 min Inactivate at 65 degrees C for 10 min note setting ~1 on grey dry black gives 65 degrees C Transformed using standard protocol ES6 UPL (unphosphatased then ligated) ES6 DL (phosphatased then ligated) ES7 UPL ES7 DL Ligation results Digested ES6 and ES7 give many transformants Note that ES6 and ES7 had a bit more DNA than other transformations since we diluted other in ligation mix and neglected to dilute ES6 and ES7 pure digests to the same cone Phosphatase appears to be working however. Ligated unphosphated ES6,7 gave many more transformants than phosphatased ligated ES6,7 Janet says this happen sometimes. Will screen 16 of each ligation. Made 16 2mL cultures in LB Amp Miniprep procedure as described in kit. Restriction digests at minipreps. Digesting all with EcoRI-HF+PstI (36x) 5uL 10x NEB4à180uL 5uL10x BSAà180uL .5uL EcoRI-HFà18uL .5uLPstIà18uL 5uL DNA 34uL H2Oà1224uL Restriction Digests placed in warm room 1.5 hour incubation 20 min inactivation at 80 degrees C DàDigest 10uL loads of digest 5uL loads of undigested ES9 Digest (also ran ES7) 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL PstI 5uL DNA 33uL H2O Digest 1 hour at 37 degrees C Kill 20min at 80 degrees C Parts came Streaked out onto Kan and Tet plates. Streakouts worked Note – tet plates dried up a bit (forgot to cover with big bin) ESA and ESB are red as expected. ES2 is purple – somewhat expected. Should not have promoter. Might be read-thru transcription) Made 2mL liquid cultures in appropriate antibiotics. Counting Colonies on L3 and L7 plates. Name/# Digested ES4 – 7 Digested ES6 – 512 L7 1:1 – 80 L7 2:4 – 247 Digest ES3 – 0 ES7 Ptest PL – 18 ES7 UPL – 204 ES6 PL Ptest – 22 L3 1:3 - 408 L3 1:3 – 218 Digested ES7 – 1280 +Control – 1280 -Control – 0 ES6 UPL – 194 L3 1:1 - 52 L3 2:4 – 600 Transformation with heat shock of RFP (it is in the PSBK3 backbone) Thaw cells on ice Aliquot 50uL Add 2uL DNA Incubate on ice 30min Incubate at 42 degrees C Incubate on ice for 2min Add 500uL LB+IPTG Incubate 1 hour at 37 degrees C on shaker (3hr) Spread 100uL onto plates Pellet leftover resuspend in 100uL and spread onto plates Also received TrpR containing plasmid Transformed using standard protocol Plated 35uL, 15uL into liquid. Backbones (calculations for ptrpL) PSBIC3 50uL at 25ug/uL We will digest ½ ugà20uL Digest mix 5uL NEB4 10x 5uL BSA 1uL EcoRI-HF 1uL PstI 20uL DNA 18uL H2O Incubate at 37 degrees C, kill at 80 degrees C Oligo Hybridization Rxn mix 3uL 100um sense oligo 3uL 100um antisense oligo 3uL PNK or T4 ligase buffer 3uL .5M NaCl Put at 95 degrees C heat block for minutes kill power on heat block, allow to cool to room temperature Ligation Nucleotide ~660 daltons/pail PSBIC3 is 2070ut=1370000g/mol Digest had .5ug/50uL*mol/1370000g=7.3*10^-6 umol/uL The hybridization should have 10uM=.01umol/uL Want ~1:4 vector:insert So 1uL vector~7.3*10^-6 We then want 4*7.3*10^-6 umol insert = 2.92*10^-5 which is .00292uL too small Dilute insert to 1:100 for final conc of 1*10^-5 umol/L, will also try 1:10 dilution, then use 3uL Ligation mix (L4) 20uL rxn volume 2uL 10x T4 buffer 1uL vector digest 3uL diluted insert 1uL T4 ligase 13uL H2O 10 minute room temp incubation 10 minute kill at 65 degrees Càthen standard transform Plates from yesterday RFP transformation K-control conc 0 RFP conc ~150 (very small colonies) K+control conc ~500 K-control 0 RFP 3 very small colonies K+control ~300 TrpR transformation Amp-Control 1 trpR 5 (2large with satellites,rest small) Amp+control a lot Ligation 4 40 colonies in all plates (including +control) After continued incubation +control shows ~50 colonies, rest stil blank Liquid cultures Biobricks ES2 no growth, restreaked from mail tube ESA growth (brown) ESB growth (red) ES5 growth trpR A+control growth A-control no growth trpR growth will miniprep ESA, ESB, ES5, and trpR Will retransform ligation 4 with heat shock and all Digest of todays minipreps (4) will use EcoRI + PstI 50uL rxn vol (x5) 5uL 10x NEB4à25uL 5uL10x NBSAà25uL 1uL EcoRi-HFà5uL 1uL PstIà5uL 10uL DNA 28uL ddH2Oà140uL Transformation Throw comps on ice Aliquot 50uL Add 2uL DNA Incubate on ice 30in Incubate at 42 degrees C 45 sec Incubate on ice 2min Add 500uL LB Incubate 37 degrees C 1hr Plate 300uL (not enough plates for spin down) 6/22 trpR-amp – streakout the big colonies and start new liquid culture ESB – inducible Kan – looks fine ES5 - inducible Kan- start liquid culture in LB-Kan- ESA tet – start liquid culture 6/23 Transformation results – L4 Ch1-control 0 Ch1+control 1000+ Annealed ptrpL 0 Digested pSBIC3 0 L4 1:10 1 L4 1:100 15 ß2 liquid cultures made Es2 (re)streak looks good not dried out Es2 liquids did not grow again, going to try multiple concentrations of tet Stock 5mg/ml previous ones were 100x dilation=50ug/mL, going to try 50ug/mL, 25ug/mL, 12.5 ug/mL 6.25 ug/mL, 3.125 ug/mL Digest 50uL volume for each of the 7 digersts (x8) 5uL 10x buffer 4à40uL 5uL10x BSAà40uL 28uL H2Oà224uL 1uL enzyme1 EcoRIà8uL 1uL enzyme PstIà8uL 10uL DNA in each digest trpR1, trpR2 RFP, ESA1, ESA2, ES5-1, ESA5-2 6/24 Miniprepped ES2(12.5x), 2xL4, RFP, ES5-1, ES5-2 TrpR Oligo Primers were received in the mail Notes on previous nightsw cultures – Both L4’s grew ES2 100x did NOT grow ES2 (12.5x) grew Es2 50x did NOT ES2 6.25x grew ES2 25x did NOT RFP grew ES5-1 grew ES5-2 grew 6/27 Digestion of MPS – RFP ES51, ES52, ES5, L4, L4, ES212.5 Master mix 500uL volume for each of the digests (7x) 5uL 10x Buffer 4à35uL 5uL 10x BSAà35uL 33uL ddH2Oà231uL 1uL enzyme EcoRIà7uL 1uL enzyme PstIà7uL 10uL DNA of each MP Notes – L4 miniprep looks too large Used filtered tip to accidentally to take up, soul mix was stuck lost 1/7th of mix Added leftover mmix to weird L4 6/28 PCR L4 (2.2x) 5uL 10x thermopolà11uL 1uL 40mM dNTPà22uL .5uL 100uM VF2à1.1uL .5uL 100uM VRà 1.1uL 1uL plasmid 1uL ventà2.2uL 1uL MgSO4à2.2uL 40uL ddH2Oà 88uL Miniprepped  trpR cultures (5mL) Digest trpR MP with PstI 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL PstI 10uL DNA 29uL ddH2O 37 degrees C for 2 hours, 80 degrees C for 20 min PCR ES5 (2.2x) (2.2x) 5uL 10x thermopolà11uL 1uL 40mM dNTPà22uL .5uL 100uM VF2à1.1uL .5uL 100uM VRà 1.1uL 1uL plasmid 1uL ventà2.2uL 1uL MgSO4à2.2uL 40uL ddH2Oà 88uL Digest of ES7 ES7 digested with XbaI, EcoRI 50uL rxn vol 5uL 10x NEB Buffer 4 5uL 10x BSA 1uL enzyme1 1uL enzyme2 5uL DNA 33uL ddH2O 2hr incubation at 37 degrees C, 20 min incubation at 80 degrees C Took out 6uL saved in UPL tube Added 5uL phosphatase buffer, 1uL phosphatase and incubated at 37 degrees C for one hr, killed at 80 degrees C for 20 min L4 results still unclear. Going to digest L4 with just EcoRI and run one gel with digested pSBIC3. Hope to see size shift. 50uL rxn vol 5uL 10x NEB4 5uL10x BSA 1uL EcoRI-HF 5uL DNA 34uL ddH2O 37 degrees C for 1hr, 20 minutes at 80 degrees C trpR PCR primers melting pt3 – forward 59.7 degrees C, reverse 47.1 degrees C thermocycler settings 95 degrees C 2min (30cycles of next 3) 95 degrees C 30sec 45 degrees C 30sec 74 degrees C 30sec 74 degrees C 5 min 4 degrees C hold Rxn mix 50uL rxn vol 5uL 10x thermopol buffer 1uL 40mm dNTP 5uL 10uM F primer 5uL 10uM R primer 1uL plasmid 1uL MgSO4 1uL vent 31uL ddH2O ES5 PCR digest Just going to use ES5-1 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL SpeI 1uL DynI 10uL PCR product 27uL ddH2O 1hr incubation at 37 degrees C, 20min incubation at 80 degrees C Going to cut L4 with SpeI (both PCR and MP) 50uL rxn vol (2.2x) 5uL 10x NEB4à11uL 5uL 10x BSAà11uL 1uL SpeIà2.2uL 5uL DNA 34uL H2Oà74.8uL 6/29 ES2 Testing Cut eith XbaI, PstI,XbaI+PstI XbaI, PstI 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL enzyme 5uL DNA 34uL water xbaI+pstI 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL XbaI 1uL PstI 5uL DNA 33uL ddH2O ES1 cut Eco Spe 50uL rxn vol 5uL NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL SpeI 5uL DNA 33uL ddH2O L6 20uL rxn vol 2uL 10x T4 ligase buffer 1uL T4 ligase 1/1/1/2uL vector 1/2/4/1uL insert 15/14/12/14uL ddH2O Controls – phosphatased ES7, unphosphatased (3uL DNA,14uL H2O) 10 min room temp, 15 min at 80 degrees C, transformed using standard procedure. Ran ES7+ES5 digest controls separately trpR PCR product digestion going to digest with EcoRI PstI and DpnI 50uL rxn mix 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL PstI 1uL DpnI 10uL DNA 27uL H2O pSBIA3 (linear) digestion 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uLPstI 1uL DpnI 20uL DNA 17uL ddH2O L9 (not in ppt yet) and trpR ligation L10 (trpRàpSBIA3) 20uL rxn volume 2uL 10x T4 Ligase buffer 1uL T4 Ligase 1uL vector 1uL insert 15uL ddH2O ES1-insert ES6- vector Transformed using standard protocol 6/30 Transformation results – Control+ - 488 ES1 – 164 +Control  - A lot ES7 UPL – 109 w/ satellites ES7 PL – 17+10satellites L6 1:1 – 20 L6 2:1 – 10 ES7 – 30 L6 1:2 – 12+2satellites L6 1:4 – 7+1satellite L9 1:1 – 35 L9 1:3 – 264 YSB143 Digest – 7 ES7 Digest – 8 ES5 Digest – 8 ES5 Digest – 0 L TrpR 1:1 – 2 -control – 0 LtrpR 1:3 – 3 -control 0 -control 2 +control – a lot+many satellites L4 restriction Test – 50uL rxn vol (3.3x) 5uL 10x NEB4à16.5uL 5uL10x BSAà16.5uL 1uL SpeIà3.3uL 10uL DNA 29uL ddH2Oà95.7uL 7/1 L6 1:1 and L9 1:1 removed from incubator and put in fridge Standard minprep procedure used. LtrpR –A-F all had growth present. Miniprepped DNA stored in freezer. 7/28 PCR L81 50uL rxn vol 25uL 2x phusion mmix 2.5uL VF2 primer 2.5uL VR primer 1uL DNA 19uL ddH2O 8/15 PCR ES9, T1, L11, trpR 50uL rxn vol 25uL 2x phusion 2.5uL VF2 primer 2.5uL VR primer 1uL DNA 19uL H2O ES1, L5, L8 50uL rxn vol 5uL NEB4 5uL 10x BSA 5uL DNA 33uLddH2O 1uL enzyme 1 1uL enzyme2 L11 trpR, T1, ES9 PCRs digestion 5uL NEB4 3uL 10x BSA 10uL DNA 1uL DpnI 1uL enzyme 1 1uL enzyme2 27uL H2O Made glycerol stocks of CFP, GFP, minipreppred CFP, GFP, retransformed L11 using standard protocol. 8/16 Redo L5 and L8 digestions. Phosphatase L5 and L8 50uL rxn vol 5uL phos. Buffer 1uL phosphatase 44uL DNA Ligation L12 and L17 20uL rxn vol 2uL T4 ligase buffer 1uL backbone 3uL insert 1uL T4 Ligase 13uL H2O Transformed L17,L12 Ligation trpR in pSBIC3 20uL rxn vol 2uL T4 ligase buffer 5uL backbone 3uL insert 9uL H2O 1uL ligase Transformed L19 8/17 Miniprepped L11 A-B Plac, L18 digestion 50rxn vol 5uL NEB4 5uL 10x BSA 5uL DNA 33uL ddH2O 1uL enzyme1 1uL enzyme2 Transformation results – L8 DSP – 2 L11 DDXP – 0 L17 – 2 L5 DSP – 2 L5 DSP – 19 +Amp – 0 T1 DDXP – 0 -amp – 0 L12 – 0 trpR ch1 – 0 pSBIC3 – 0 trpR digest – 0 cultured 2 L17 colonies 8/22 Zipper PCR 50uL rxn vol 2.5 uL F primer 2.5uL R primer 1uL DNA 25uL phusion 19uL H2O L12 test digest 5 and one ES1 digest for L19 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL enzyme1 1uL enzyme 2 10uL enzyme 28uL ddH2O 8/23 PCR GFP 50 rxn vol 25uL phusion 2.5uL GFP MF 2.5uL MR 1uL 19uL Ligations trpR+pSBIC3, L19, ES9+ES1, L4+L18, L11+plac 20uL rxn vol 2uL T4 ligase buffer 1uL backbone 3uL insert 1uL T4 Ligase 13uL dH2O Ligations – L19 vector ES1, Insert ES9 710 – insert 710nzipper, vector psBIC3 711 – insert 711czippe, vector pSBIC3 8/24 Transformations L19 – 4-5 L4+L18 – 3 +amp ES7/+Amp – a lot pLac+L11 – 0 -control (all) – 0 710 711 +ahl? L3 and T1 digestions – L3 and (DES) 50rxn vol 5uL NEB4 5uL 10x BSA 10uL DNA 1uL DpnI 1uL EcoRI 1uL SpeI 27uL ddH2O T1 50uL rxn vol 5uL NEB4 5uL 10x BSA 5uL DNA 1uL EcoRI 1uL XbaI 33uL ddH2O L22 Ligations (L3+T1) 20uL rxn vol 2uL T4 Ligase buffer 1uL backbone 3uL insert 1uL T4 ligase 13uL ddH2O Miniprepped L4+L18, L19A,B 8/25 Note – L4+18 cultures are noticeably red. (good) Just going to send everything for sequencing. Not much to gain from gel. Started cultures of L12A, trpR pSBIC3, L12A, L710A-B, L711A-B, L3-3, plae+L11AB, trpRC Miniprepped cultures Test cats – (11x) 25uL rxn vol 2.5uL 10x NEB4à27.5uL 2.5uL 10x BSAà27.5uL 1uL EcoRI-HFà11uL 1uL PstIà 11uL 5uL DNA 13uL ddH2Oà143uL 8/30 GFP round the horn Mutagenesis ligation 20uL rxn vol 2uL 10x T4 ligase buffer 1uL T4 ligase 2uL DNA 15uL ddH2O L3-3 digestion 50uL rxn vol 5uL NEB4 5uL 10x BSA 5uL DNA 1uL SpeI 1uL PstI 33uL ddH2O L22 Ligation 20uL rxn vol 2uL 10x T4 ligase buffer 1uL backbone L3 3uL insert T1 1uL ligase 13uL ddH2O Cultured L19A-C, L22, transformed L22, GFP 8/31 GFP/L22 transformations -control – 0 GFP – a lot L22 – 42 Miniprepped L22B,C, arbitrarily did not miniprep A, put in fridge. 9/8 OBS – subculture of DH3OC comp cells wuth different concs of parent culture 500L/5mL 1000L/5mL Incubation time – 17hrs Aronch period should be in the log phase to dilute stock to 1:100. Diluted stock should be grown from morning. 9/12 711 pCR CZ 50uL rxn vol 25uL phusion 2.5uL F primer 2.5uL R primer 1uL DNA 19uL dH2O GFP miPCR 50uL rxn vol 25uL phusion 2.5uL GFP MF 2.5uL GFP MR 1uL DNA GFPmi 19uL ddH2O L3-3 PCR 25uL phusion 2.5 VF2 2.5uL VR 1uL DNA 19uL ddH2O 41PCR 25uL phusion 2.5uL VF2 2.5uL VR 1uL DNA 19uL ddH2O GFPlm PCR 25uL phusion 2.5uL VF2 2.5uL VR 1uL DNA 19uL ddH2O L3, L11 PCRs purified L3, L11 digests (going to gel extract so DpnI unnecessary) L3 – EcoRI-HF, SpeI L11 0 XbaI, PstI 5uL 10x NEB4 5uL 10x BSA 1uL enzyme 1 1uL enzyme 2 20uL DNAßsince we are gel extracting 18uL ddH2O 8/13 GFPml, PCR, 711 PCR DD purified 711 PCR DD purified with EcoRI-HF, PstI (to ligate into pSBIC3) 5uL 10x NEB4 5uL 10x BSA 1uL Fzce 1uL Pst 10uL DNA 28uL ddH2O L3,L11 digests killed at 80 degrees C ES4-ES1 sent for sequencing. (all of today’s tubes put in green rack) Miniprepped ES1 711 digest killed at 80 degrees C Comp cellsàsee protocol on OWW Tfse of comp cells Place on amp is used as a test Black mark is second half of tube that was repelleted, may effect competency Amp placA+Amp placB LB plNCA+NCB=negative control Ligations L22 insert L3 Backbone T1 20uL rxn vol 2uL 10x T4 buffer 1uL backbone 3uL insert 1uL T4 ligase 13uL ddH2O Plate L11, insert L11, backbone plae Transformed with janet’s cells using standard protocols 9/14 Started 2 cultures each of 711, L22 Streaked out (poorly)plae+L11 9/15 Miniprepped 711, L22 cultures Restreaked plae+L11 GFP nzipped digests GFPmiPCR 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL AgeI-HF 1uL EcoRI-HF 10uL DNA 1uL DpnI 27uL ddH2O NZipper 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL NgoMIV 10uL DNA 28uL ddH2O GFPml Nzipper ligation (NZGFP) Insert GFPml Backbone 710A 20uL rxn vol 2uL 10x T4 ligase buffer 1uL 710A 3uL GFPml 1uL T4 ligase 13uL ddH2O Restreaked plae+L11 Transformed NZGFP using quick protocol L12 Digestion 50uL rxn vol 5uL 10x NEB4 5uL 10x BSA 1uL EcoRI-HF 1uL XbaI 10uL DNA 28uL ddH2O Order ES1 and retransform ES1 Send ES9 sequences to Vershon Redesign lovtap mutagenesis primers Write up adder experimental part Miniprep plac+L11 PCR plac+L11A 25uL phusion 2.5uL VF2 2.5uL VR 1uL DNA 19uL ddH2O PCR purify L22 PCR and L12ADEX L22 Digestion DDES 5uL NEB4 5uL 10x BSA 10uL DNA 1uL DPN1 1uL EcoRI 1uL SpeI 27 ddH2O L4+L18 digestion DEX 5uL NEB4 5uL 10x BSA 1uL EcoRI 1uL XbaI 10uL DNA 28uL ddHwO PCR purify L4+L18 and plac+L11 PCR Plac+L11 digestion DDES 5uL NEB4 5uL 10x BSA 1uL EcoRI 1uL SpeI 10uL DNA 1uL DPNI 27uL ddH2O Ligate L22+L12 2uL 10x T4 ligase buffer 1uL backbone L12 3uL insert L22 1uL ligase 13uL ddH2O L21 transformation/colonies -control – 0 +control – a lot L21 – 0 L22 DDES – 0 L12 DEX – 5 9/19 Miniprepped ES1A,B Culture plac/L11+L4/L18A,B, L21A-B 9/20 Miniprep all cultures made on 9/19 9/22 NZGFP ligation 20uL rxn vol 2uL 10x T4 ligase buffer 3uL 710A 9uL GFPml 1uL T4 ligase 5uL ddH2O L21 Backbone L12DEX Insert L21 DDES (S/T) 20/20uL rxn vol 2/2uL T4 buffer 1/2uL backbone 6/2uL insert 1/1uL T4 ligase 10/13uL ddH2O Started new cultures of ESB, RBS lovtap term. Promoter RBS lovtap term. 9/23 Miniprepped cultures from 9/22