Team:UChicago/Notebook
Notebook
6/14/18 -Got familiar with lab -Made LB and normal YPAUD liquid media -Labelled liquid media and left on bench shelf -Make chloramphenicol plates -250mg chloramphenicol; marked by CAM 6/15/18 -Made Arg- YPAUD liquid media (-Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM (1.7g of CSM -Arg -Ura, 1.7g of uracil) -Marked by 3 blue stripes -Made Arg- YPAUD plates -Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM -Marked by three blue stripes -PCR amplify arg-ars sequence -Total volume: 50µL -35µL water -10µL 5X phusion buffer -1µL dNTPs -2.5µL 10µM primer stock (forward and reverse arg-ars primers) -1µL pOW1 (17.6 ng/µL) DNA stock (0.8µL DNA, 4.2µL H2O) -0.5µL phusion -Stored in refrigerator next to PCR machine (labeled iGEM arg-ars PCR) 6/18/18 -Gel electrophoresis of arg-ars sequence (PCR amplified on 6/15/18) -Check if PCR was successful -Agarose gel: -40 mL 1X SB buffer, 0.32g agarose -Mixed and microwaved for 35 seconds -Added 2µL EtBr to agarose gel in flask -Agarose gel opaque and gray when fully polymerized -Sat in chamber, completely covered in buffer, until ready to run the gel -Gel was unsuccessful, neither the ladder band nor the PCR product band could be observed -Suspected problem: samples not loaded into wells properly -Large smear of DNA across wells observed -PCR amplified arg-ars sequence with 1 min per Kb (5 min since Arg-ars sequence is 4.86 Kb) -Labeled in blue marker 6/19/18 -PCR amplified arg-ars sequence with 30 seconds per Kb (2.5 minutes since Arg-ars sequence is 4.86 Kb) -Labeled in black marker -Gel purification Arg-ars PCR products -Made 0.8% agarose gel -0.32 g agarose; 40 mL 1X SB buffer; 1µL EtBr -Each well has 25µL of sample -Well 1 loaded with 1 min per Kb arg-ars PCR product -Well 3 loaded with DNA ladder--- -Well 5 loaded with 30 sec per Kb arg-ars PCR product -Ran at 120V for 25 min (4.86 Kb sequence) -Well 5 came out clear, however wells 1 and 3 were diffuse to be observed -Suspected problem: holding micropipette to the bottom of the well when pushing to the second stop, when pulling the micropipette out, dye doesn’t remain 6/20/18 -Gel purification Arg-ars PCR products -Made 0.8% agarose gel -0.32 g agarose; 40 mL 1X SB buffer; 1µL EtBr -Well 1 loaded with 1 min per Kb arg-ars PCR product -Well 4 loaded with DNA ladder -Well 7 loaded with 30 sec per Kb arg-ars PCR product -Ran at 120V for 25 min (4.86 Kb sequence) Bands were smeared -Michael: voltage likely too high, run at 90V; ladder lane did not have clear bands, try running the ladders against each other; the PCR product with 30 seconds per Kb produced very faint bands, use the protocol with 1 min per Kb -Identified the (large and rather smeared) region where the arg-ars sequence was likely to be and excised it -Placed in eppendorf tube and added 450 µL of GQ buffwe -Placed in 50º bath for 10 minutes -Added 450µL of GQ buffer (solubilization buffer), centrifuged at 5.0k rpm for 1 min -Added 200 µL of PE buffer (wash buffer), centrifuge at 13.0k rpm for 30 seconds -Added 200µL of PE buffer (wash buffer), centrifuged at 13.0k rpm for 3 minutes -Inverted and set to dry for 3 minutes -Nanodrop detected no DNA (no peak at wavelength of 260 nm) -PCR amplified arg-ars sequence (1 min per Kb) -Made two PCR products (each 50µL) -Used protocol outlined above -Cleaned lab area 6/21/18 -Gel purification of arg-ars PCR product -0.8% agarose gel with 1X SB buffer, 2µL EtBr -Run at 105V for 30 minutes -Lane 1: arg-ars PCR product; lane 3: 100 bp ladder -Bands observed in gel were too smeared and also too short to be the desired sequence -Simone’s suggestions -Not trying to PCR the entirety of POW1 (which is 4.86 Kb), only trying to amplify a 2.5 Kb section that contains ScARG4 -Run PCR with annealing temp at 58ºC instead of 55ºC 6/22/18 -PCR amplified arg-ars sequence with an annealing temp of 58ºC -Gel electrophoresis of arg-ars PCR product -0.8% agarose gel with 1X SB buffer and 2µL EtBr -105V for 35 minutes -Lane 1: 1 Kb ladder; lane 3: arg-ars PCR product; lane 5: arg-ars PCR product -Lane 5 did not come out clear -Ladder too bright, added too great a volume to the well (20 µL instead of 5µL) -Gel purify arg-ars PCR product -No DNA detected by the nanodrop 6/22/18 -Did diagnostic work on the PCR reaction performed by RF -Suspected cause 1: Template was at 18.2 ng/µL instead of the 96.5 labeled on the tube -Sample also has a 260/230 of 1.27 indicating EtOH contamination inflating this value -Likely that there is not enough template to successfully create PCR product -Only 2µL remain anyway, so need new pOW1 to proceed anyway -Suspected Cause 2: Primers are not specific enough. Forward primer’s last 5 3’ bases match to 7 locations. One of these forms a 600 bp product that is consistent with the lower band. Can extend the primer 5 bases to create a specific primer that only binds to its target site. -To grow more pOW1, prepared an overnight culture of PPY12 as Allison instructed the plasmid to be grown in yeast 6/23/18 -Talked with Dr. Glick -Indicated that cannot grow more plasmid in the PPY12 overnights as they need to be grown in bacteria -Transformed 1µL of remaining pOW1 plasmid into DH5α cells. Plated onto an LB+Amp plate (See below) -Created 20 LB+Amp plates following Glick lab plate recipe. Plated both my sample on 1 and a negative control on another. Did not have a strain that I knew had Amp resistance already, so I was unable to do a complete positive control. 6/24/18 -LB+ Amp Plate result -Negative control had no growth indicating the antibiotics are in sufficient quantity to kill non antibiotic resistant bacteria -pOW1 sample had too many colonies to count indicating a successful transformation was likely (Had no positive control, so cannot be fully certain until attempt to isolate the plasmid is performed) -Inoculated 5 overnight liquid cultures (4 for plasmid harvesting and one to make a glycerol stock and future positive Amp control) 6/25/18 -Miniprep Liquid cultures A→ D -Concentrations -A: 748.9 ng/µL -B: 573.3 ng/µL -C: 527.9 ng/µL D: 554.2 ng/µL -Made glycerol stock of Liquid culture E 6/26/18 -PCR amplified arg-ars sequence from pOW1 samples B-D -Want ~20 ng of the plasmid DNA (these dilutions result in pOW1 concentrations of 20 ng/µL) A: 0.8µL pOW1 A, 29.2 µL H2O -B: 0.8µL pOW1 A, 23.0 µL H2O -C: 0.8µL pOW1 A, 21.1 µL H2O -D: 0.8µL pOW1 A, 22.2 µL H2O -Made glycerol stock of liquid pOW1 culture E -Gel electrophoresis of arg-ars pOW1 A-D PCR products -Lane 1: 1 Kb ladder; lane 2: pOW1 B; lane 4: pOW1 C; lane 6: pOW1 D -Ran at 110V for 30 minutes -Gel imaging showed only a smaller band around 100 bp for lane 2 (pOW1 sample B) -Suggests primer issue; will address in team meeting tomorrow -PCR amplified arg-ars sequences from pOW1 sample A -PCR amplification of pOW1 sample B side by side with CC -RF: followed previous PCR protocol, used arg-ars F1 and R1 primers -CC: Followed NEB Phusion protocol found on their website. Is as follows: -10µL of 5x Phusion HF Buffer -1µL 10mM dNTPs -2.5µL of Forward Primer -2.5µL of Reverse Primer -1µL of Template DNA (Diluted 50X from original stock) -0.5µL of Phusion Polymerase -Annealing temperature was decreased to 57ºC -Gel electrophoresis of pOW1 B with CC -Lane 1: 1 Kb ladder -Lane 2: RF PCR protocol with primers F1 and R1 -Lane 4: CC pOW1 B sample -This was unnecessary, it is the entire plasmid, not a PCR product -Lane 6: CC -This is Cian’s new PCR ‘recipe’ with F2 and R2 -The new primers Simone gave us -Gel imaging showed that the PCR was unsuccessful -We might not have the pOW1 plasmid -Ran restriction digest of pOW1 samples A-D and last year’s stock with Xbal1 -10µL reaction -1µL cutsmart buffer -1µL of each DNA sample including last year’s stock (which we know to be pOW1) -0.1µL of Xbal1 GQ -7.9µL of milliQ water -Place reaction in 37ºC incubator for one hour -Ran gel electrophoresis of the five 10µL reactions -Lane 1: “Go Green” 1 Kb ladder from Simone -Lane 3: control pOW1 sample (from 2017) -Lane 4: pOW1 sample A -Lane 6: pOW1 sample B -Lane 7: pOW1 sample C -Lane 8: pOW1 sample D -Lane 10: 1 Kb ladder from our -20ºC freezer (below benchtop) 6/27/18 -Ran PCR of pOW1 protocol -Annealing temperature of 62ºC -Reaction 1: F1 R1 -Reaction 2: F1 R2 -Reaction 3: F2 R1 -Reaction 4: F2 R2 -Protocol: 50µL PCR product -32.5 µL milliQ water -10µL of 5x Phusion HF Buffer -1µL 10 µM dNTPs -2.5µL of Forward Primer -2.5µL of Reverse Primer -1µL of Template DNA (Diluted 50X from original stock) -Used pOW1 sample B -0.5µL of Phusion Polymerase -PCR product 4 opened in the machine; no product remaining -Ran gel of pOW1 sample B PCR products -Lane 1: “Go Green” 1 Kb ladder -Lane 3: 1 Kb ladder found in -20ºC freezer below benchtop -Lane 5: pOW1 PCR product 1 (F1 + R1) -Lane 7: pOW1 PCR product 2 (F1 + R2) -Lane 9: pOW1 PCR product 3 (F2 + R2) -110V for 30 minutes -Results of gel -Our 1 Kb is accurate and had bands comparable to the “Go Green” 1 Kb ladder provided by Simone (grad student) -Sterilized new eppendorf tubes in autoclave -Dry cycle, 15 minutes -Sent 4.5µL of mini prepped pOW1 plasmid sample, 3µL of seq.F.3 primer, and 3 µL of seq.R.3 primer for sequencing -Made a 1:10 dilution of the stock primers -Made a 3:10 dilution of the stock pOW1 plasmid sample C -To result in a concentration of roughly 300 ng/µL -PCR amplified reaction 4 again -F2 and R2 6/28/18 -Gel electrophoresis of reaction 4 -PCR product from 6/27/18 with annealing temperature of 62ºC -Ran at 110V for 30 minutes -Lane 1: “Go Green” 1 Kb ladder -Lane 3: reaction 4 -Gel imaging showed only one DNA band which was smaller than the 2.55 Kb region we are trying to amplify -Ran PCR protocol with an annealing temperature of 56ºC -Reaction 1: F1 R1 -Reaction 2: F1 R2 -Reaction 3: F2 R1 -Reaction 4: F2 R2 -50X dilution of pOW1 sample B - --49 µL of milliQ water, 1µL sample -Gel electrophoresis and imaging of reactions 1-3 PCR products -Ran at 110V for 30 minutes -PCR tube containing reaction 4 opened so no product remained -Lane 1 & lane 8: “Go Green” 1 Kb ladder - -Suspected contents from lane 2 might have bled over into the lane 1 -Lane 2: reaction 1 PCR product -Lane 4: reaction 2 PCR product -Lane 6: reaction 3 PCR product -Gel showed only the shorter band, not the 2.55 Kb band we are looking for -Remade master mix for reaction 4 and re-ran PCR protocol -Reaction 4: F2 and R2 6/28/18 -Gel electrophoresis of reactions 1-4 -Ran at 110V 0.8% Agarose -All bands came back negative -Only had the 600bp banding.
7/2/18 -PCR of Samples A-F of pOW1 using 2-step method from last year (outlined below in pictures) -Annealing temperature 68°C -Ran out of Phusion DNA Polymerase-working on getting more -As a result, only Samples A-D could be PCRd properly -Potential issue with PCR-Step 1 of 2 step not entered correctly; may need to redo PCR but after checking results -Gel Electrophoresis -100 V ran for 20 min -1% 50 mL Gel created as per Jason’s introductory guide 7/3/18 -PCR Samples A-F or pOW1 using 2-step method BUT WITH Taq Polymerase/Buffer instead of Phusion done correctly as per photo -Annealing Temperature 68°C -Gel electrophoresis -5ul ladder, 2 ul loading dye added to each sample -Sample A not electrophoresed -1 % 50 mL Gel created as per Jason’s introductory guide -110 V 7/5/18 -Learned to use nanodrop machine -Attempted to digest pBSC13mut with Hpa1 and measure concentration, however concentration was quite low -5ul 10x Cutsmart Buffer used -.5ul Hpa1 -Template DNA added to 1 microgram -MilliQ water added to 50ul -Given ArgArs sequence by Simone, however there was contamination so PCR clean up performed -Used Wizard SV Gel and PCR Clean-Up System protocol -Still no clear usable results and thus must PCR ArgArs sequence from given DNA on our own 7/6/18 -Discovered concentration of pBSC13mut is lower than expected (17.6ng/ul); may explain why digestion with Hpa1 doesn’t seem to yield results -Began transformation of more pBSC13mut to be completed over weekend -Protocol is as follows: -1. Get DH5alpha cells from the bottom shelf of the -80C and put on Ice. -2. Once defrosted, add 1µL of the transformation product into the cells. Mix by flicking the bottom. DO NOT PIPETTE TO MIX!! -3. Incubate on ice for 30 min. -4. Heat Shock the bacteria for 45sec in the 42C water bath. Let recover on ice for 2 min -5. Add 300µL of SOC medium and place in shaking 37C incubator for 1hr -6. Plate 50µL on Plate. Place place in the 37C incubator overnight (Place after 4:00pm) -Ran PCR w/ POW1 using protocol sent by Simone -Protocol is as follows: -12.5ul Q5 Master Mix -1.25ul F and R primers each -1ul of template added -Water added to 25ul -5ul ladder, 2 ul loading dye added to each sample -Q5 Polymerase used and Q5 2x Master-Mix polymerase protocol applied from online -1% 50mL gel Created and ran at 110V -No results for band 7/7/18 -Set up overnight cultures as per Cian’s instructions so that they could be miniprepped -6 colonies chosen and labelled A-F -Cian’s Overnight cultures protocol used (insert link) 7/8/18 -Mini Prepped Varun’s overnight cultures -Concentrations ranged from 80ng/µL to 115ng/µL 7/9/18 -Prepared 100µM stocks of the Ben and Gib primers for alternates to the ArgArs -Ran PCR on pOW1 using these Primers -Used Q5 polymerase as per online protocol -12.5ul Q5 Master Mix -1.25ul F and R primers each -1ul of template added -Water added to 25ul -Ran diagnostic digest of pBSC13mut w/ Hpa1 -To 10ul rather than to 50ul -1µg of DNA added to each sample -.1ul Hpa1 added -1ul Cutsmart 10x Buffer -Water filled to 10ul -If greater than 10µl, no water added -Ran PCR of ArgArs sequence again -PCR protocol used: Igem ArgArs -5ul ladder GoGreen, 2 ul loading dye (Ran out of Go Green, new 1Kb ladder used -1% 50 mL gel Created & ran at 120V -This time, 2.55kb band did form -However, I accidentally discarded the gel itself and thus must repeat the PCR -Excised the Well 2 and Well 3 Bands from GE -Ran Gel Purification -Ended up with Well 2 concentration of 35.5ul and Well 3 concentration of 49.9ul 7/10/18 -25µL PCR product of arg-ars sequence from pOW1 plasmid - touch-down PCR -15.6µL milliQ water -5µL 5X buffer -1µL template DNA (pOW1 plasmid) -1.3µL of F2 -1.3 µL of R1 -0.5µL dNTP -0.3µL Phusion -iGEM arg-ars PCR cycle -Diagnostic digest of pBSC13mut with Hpa; 10µL product -8.8 µL milliQ water -0.1µL cutsmart buffer -1 µL of pBSC13mut samples A-F -0.1µL Hpa1 -Incubated for 1 hour at 37ºC -Gel electrophoresis of diagnostic digests run at 110V for 30 minutes -0.8% agarose gel -Well 1: Quickload 1 Kb+ ladder -Well 2: pBSC13mut A -Well 3: pBSC13mut B -Well 4: pBSC13mut C -Well 5: pBSC13mut D -Well 6: pBSC13mut E -Well 7: pBSC13mut F -Gel electrophoresis of pOW1 arg-ars PCR product run at 110V for 12:10 -Well 1: Quickload 1 Kb+ ladder -Well 2: pOW1 arg-ars PCR product with F2 and R1 -Well 3: pOW1 arg-ars PCR product with Ben Glick Primers -Labelled S1 -25µL PCR product of Stage 2 and ArgArs from yesterday's products -Recipe -15.6µL milliQ water -5µL 5X buffer -1µL template DNA ArgArs product from yesterday (Stage 1 PCR Product) -1.3µL of F2(FGib) -1.3 µL of R1(RGib) -0.5µL dNTP -0.3µL Phusion -Only 1 thermocycler was open, so created iGEM Combi Protocol to do both simultaneously -First 5 cycles are a gradient from 56 to 65. Gib rxn run at the 56 end. ArgArs run at the 65 end -Final 27 cycles run at 65 entire block (Reduce nonspecific Gib binding) -Diagnostic digest of all pSB1C3 mut stocks that I could find -Found 9 stocks from 2017 -Did 10µL diagnostic digest with each as well as two controls -Negative: pSB1C3 with HpaI (Expect no cut) -Positive: pSB1C3 with EcoRI(Expect a cut) 7/11/18 -Ran Cian’s PCR’s on one large gel -Large gel used, 0.8% agarose (120ml of SB Buffer) -SB Buffer used to keep continuity with Glick Lab’s use of SB Buffer -Well 1 Loaded with ladder 5ul -Well 2 Loaded with Stock B 20ul -Wells 3-4 Loaded with - and + Controls 20ul, respectively -Wells 6-13 Loaded with Stocks 1-8 20ul -Wells 14-17 Loaded with ArgArs amplified regular way 20ul -Wells 18-21 Loaded with Stage 2 Alternate ArgArgs amplification -Stocks 2,3,4, & 7 of pSB1C3mut resulted in bands of somewhat correct length -These must be transformed -Lots of streaking in wells 14-21, must be result of SB Buffer → Use TAE -Transformed PSB1C3 stocks 2,3,4,7 along with RFP- PSB1C3 from iGem parts and Competency cells test -Used JM109 Bacteria instead of DH5A -Process is same as listed above otherwise -Ran PCR on Simone’s Arg/Ars Sequence using Q5 Polymerase with exact same protocol as earlier trial which resulted in correct banding -25ul reaction total, 5 reactions labeled 1-5 -12.5ul Q5 Master Mix -1.25ul each of F1 and R1 Primers -9ul template -1ul H20 -0.8% 50ml Gel created to run DNA on (0.4g agarose) -Once again, TAE buffer used 7/12/18 -Removed RFP-pSB1C3 stock + pSB1C3mut stocks 2, 3, 4, and 7 from 37ºC incubator at 9:15am -Made liquid overnight cultures of pSBC13mut 3 -Picked 6 colonies; labelled A-F -Put in 37ºC incubator at 5:00pm -Began streak purification process of pSBC13mut sample RFP-pSB1C3 + pSB1C3mut stocks 2, 4, and 7 -Put in 37ºC incubator at 5:00pm -Stage 2 PCR (with Gib F and R primers); used GeneHackers cycle -Annealing temperature of 57ºC -Two 25µL reactions (labeled A and B) with Phusion -12.6µL milliQ water -5µL 5X buffer -3µL template DNA ArgArs product from yesterday (Stage 1 PCR Product) -1.3µL of F2 (FGib) -1.3 µL of R1(RGib) -0.5µL dNTP -0.3µL Phusion -2 reactions (labeled C and D) with Q5 (VP) -Ran gel of pOW1 arg-ars touchdown PCR product (56º – 70ºC) -Gel purification of pOW1 arg-ars touchdown PCR product -Eppendorf tubes containing samples E and F are likely mixed together -No gel fragment in eppendorf tube F after band excision -1µL membrane binding solution per 0.001g of gel fragment excised 7/12/18 -PCR of S1 Ben Alternate ArgArs Product -Using Q5 Polymerase as follows -12.5ul Q5 Master Mix -1.25ul GibF and Gib R each -4ul ArgArs PCR1 Product (Well 3) -6ul H20 -Labelled C and D -PCR of ArgArs 2 protrocols -ArgArs Programming (4rxm total) -5µL Phusion HF buffer -0.5 dNTP (New stock from NEB) -1.3 µL F2 -1.3µL R1 -1µL pOW1 D (50X Dilution) -15.6ul H20 -0.3µL Phusion Poly -Assembled as part of a Master mix with Gradient -Standard PCR Protocol with a gradient -Above recipe -Assembled as part of a Master Mix with above protocol -Gradient from 56C to 70C -Gel Results (Each PCR run on a seperate gel) -All 12 reactions showed amplification of a band between 2Kb and 3Kb. Indicates correct product synthesized -All gels showed banding below 500 bp. Likely a combination of mispriming and Remaining primer as it is extremely faint. -Overall impression: Desired product can be synthesized at a range of temperatures using standard protocols. Issues with cloning likely have been technique related or a problem with the dNTP stock as it was homemade. Using the new dNTP from NEB yielded correct results. -Gel Purification of all 12 fragments -Purification of ArgArs -Followed Promega protocol for Wizard PCR cleanup. Yields were less than 10 ng/µL and 260/230 was 0.03 avg. Discarded samples. Likely due to improper incubation with membrane binding solution (See reasoning below) -Purification of Gradient products -Done with RF and VP -Followed directions according to promega protocol. Measured concentrations were 30-56 ng/µL with 260/230 of 0.46. Still not great, but might be able to improve on future runs. Suspect incubation with the membrane binding solution is too low and DNA does not have sufficient time to bind to column. Could also be a problem with adding the membrane binding solution as this was a problem in my lab with the Zymo Kit. May be able to be used for Gibson but will need to overshoot 7/13/18 -Removed overnight plates of streak purified RFP-pSB1C3 stock + pSB1C3mut stocks 2, 3, 4, and 7 -Only plates pSB1C3-2 and pSB1C3 4 had colonies -Other plates had no observable growth -Mini-prepped overnight liquid bacterial cultures of pSB1C3mut-3 -Followed manufacturer's instructions for the PureYield Plasmid Miniprep Promega Kit -Got less than ideal results when nano-dropped -Ran an ethanol precipitation protocol for the mini-prepped pSB1C3mut-3 DNA -Didn’t work, no pellets seen in the eppendorf tubes -Made overnight cultures of pSBC13mut 2, 3, and 4 -Full digest of D and E -32.2µL of nuclease free water -5µL of buffer J -12.3 µL of D and E -0.5µL of Hpa1 -Placed overnight liquid culture tubes and plates in 37ºC incubator at 6:00pm
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