Team:CU-Boulder/Experiments
MG 31.5 ul: dH2O (water is added to make the reaction mixture up to 50ul) 10 ul : 5X phusion HF 2.5 ul: 10 um FWD primer 2.5 ul: 10 um REV primer 1 ul: MgCl 1ul: new plasmid 1 ul: 10mM dNTPS 0.5 ul: Phusion hot start flex DNA polymerase MG and DMSO 30 ul: dH2O (water is added to make the reaction mixture up to 50ul) 10 ul : 5X phusion HF 2.5 ul: 10 um FWD primer 2.5 ul: 10 um REV primer 1 ul: MgCl 1.5ul: DMSO 1ul: new plasmid 1 ul: 10mM dNTPS 0.5 ul: Phusion hot start flex DNA polymerase DMSO 31 ul: dH2O (water is added to make the reaction mixture up to 50ul) 10 ul : 5X phusion HF 2.5 ul: 10 um FWD primer 2.5 ul: 10 um REV primer 1.5 ul: DMSO 1ul: new plasmid 1 ul: 10mM dNTPS 0.5 ul: Phusion hot start flex DNA polymerase NOTHING 32.5 ul: dH2O (water is added to make the reaction mixture up to 50ul) 10 ul : 5X phusion HF 2.5 ul: 10 um FWD primer 2.5 ul: 10 um REV primer 1ul: new plasmid 1 ul: 10mM dNTPS 0.5 ul: Phusion hot start flex DNA polymerase Ran at: 98---30 sec (35 cycles) 98---10 sec 60-72-- 30 sec 72--- 2:30 minutes ----------------------------------------- 72---10 minutes 4---infinite
1. Turn on bath and place SOC in bath 2. thaw cells on ice 3. 10ul of competent cells into tube 4. 1 ul of assembled product to cells 5. mix by pipet or flicking the tube 6. place on ice for 30 minutes 7. heat shock in bath at 42 degrees for 30 seconds 8. put back on ice for 2 minutes 9. add 190 ul of SOC media 10. place in 37 degree incubator on shaker for 60 minutes 11. spread on plates 12. incubate at 37 degrees overnight
10ul of Gibson Assembly Master Mix (2X) 10ul-x of dH2O Xul of vector + Xul of insert (0.02-0/5 pmols) Tota volume: 20ul Incubate at 50 degrees for 15 minutes Store at -20 until use
1. Pick a colony, circle and label it 2. Prepare the LB broth in a 10mL tube, pour 5mL of LB into the tube 3. Add 5ul of necessary antibiotic to the culturing tube 4. Select colony by using a sterile toothpick or pipet tip, gentilly pick it up 5. Place colony into culture tube and incubate at 37 degrees overnight
1. Weigh out 0.5g of Agarose powder, place in flask 2. Add 50ul of TAE to flask, swirl solution 3. Cover flask with a paper towel, use tape to hold it in place 4. Microwave for about 1.5 minutes 5. Add 3ul of EtBr OR 5ul of SYBR Green dye 6. Pour into mold and let set for about 15 minutes
1. Warm plates in incubator 2. Thaw BL21 (protein competent cells) on ice for 10 minutes 3. Add 1ul of plasmid to the cells, fix to mix 4. Place solution on ice for 30 minutes 5. Heat shock at 42 degrees for 10 seconds 6. Place back on ice for 5 minutes 7. Pipette 95ul of room temperature SOC 8. Place on shaker and incubate at 37 degrees for 1 hour 9. Plate 100ul of cells on each plate 10. Incubate at 30 degrees for 24-36 hours
1. Select a colony and label it 2. Prepare LB, add 10ml of LB broth and 10ul of select antibiotic 3. Pick up colony and resuspended in culture tube 4. Incubate at 37 degrees until OD600 reaches 0.4–0.8 5. Induce with 4 or 40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) and induce for 3 to 5 hours at 37°C. 6. Check on gel
For a 50ul post-PCR solution Add 5ul of cutsmart buffer to 50ul post-PCR reaction Add 2ul of DPN1 enzyme Incubate solution for 1 hour at 37 degrees PCR cleanup to stop enzymatic reaction
1. Gels electrophoresed with MOPS or MES running buffers must be prefixed with a 50% methanol and 7% acetic acid solution for 15 minutes and then washed with ultrapure water to remove fixing solution. GelCode Blue Stain penetrates prefixed gels better than non-fixed gels and, therefore, standard SDS-PAGE gels may also be prefixed with good results. 2. Wash SDS-PAGE or native PAGE gels as follows: • SDS-PAGE: After electrophoresis, place gel in a clean tray and rinse 3 × 5 minutes with 100-200mL of ultrapure water. Alternatively, wash gel in 1-2L of ultrapure water with gentle shaking for 15 minutes. 3. Mix the GelCode Blue Stain Reagent solution immediately before use by gently inverting or tipping and swirling the bottle several times. Do not shake bottle to mix the solution. 4. Add 20mL of GelCode Blue Stain Reagent for an 8 × 10cm mini gel. Use more reagent as needed if using a large tray. Gently shake tray and periodically monitor protein band development. Stain intensity reaches a maximum within approximately 1 hour. Gels may be stained overnight without increasing background. 5. To destain (water wash enhancement step) gel, replace Stain Reagent with ultrapure water. For optimal results, change water several times for 1-2 hours. This step enhances stain sensitivity, as weak protein bands continue to develop.
Procedures completed using Qiagen ordered kits.
1. Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. VWR #60818-667) at room temperature. Place SOC recovery medium in a 37°C water bath. Pre-warm selective plates at 37°C for 1 hour. 2. Place electroporation cuvettes (1 mm) and microcentrifuge tubes on ice. 3. As a positive control for transformation, dilute the control pUC19 by 1:5 to a final concentration of 10 pg/μl using sterile water. Heat-denatured ligation reactions can be used for electroporation directly; however, column purification is recommended. 4. Thaw NEB Turbo Electrocompetent cells on ice (about 10 min) and mix cells by flicking gently. Transfer 25 μl of the cells (or the amount specified for the cuvettes) to a chilled microcentrifuge tube. Add 1 μl of the DNA solution. 5. Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette. Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 2.1 kV, 100 Ω, and 25 μF. The typical time constant is ~2.6 milliseconds. 6. Immediately add 975 µl of 37°C SOC to the cuvette, gently mix up and down twice, then transfer to the 17 mm x 100 mm round- bottom culture tube. 7. Shake vigorously (250 rpm) or rotate at 37°C for 1 hour. 8. Dilute the cells as appropriate then spread 100-200 μl cells onto a pre-warmed selective plate. 9. Incubate plates 8 hours to overnight at 37°C. set machine to 1.8V place cuvette in machine press both pulse buttons for 3 seconds, you should hear a beep after If you hear a pop or see a flash your culture has arched. This means one of a few things, your cell concentration is too high, dilute with 10% glycerol. Your DNA is not pure, either used less or use another DNA stock. Or both of the previous issue is present. Add 600ul of LB to the cuvette (or 250ul of SOC) and pipette up and down, take out some culture and grow on a shaker for 1 hour Place culture on selective plate