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Revision as of 03:56, 14 October 2018
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.
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