Difference between revisions of "Team:UChicago/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

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