Agar Plates + Calculations

Transcription

1 Agar Plates + Calculations Thursday, May 25, :27 AM LB media prep with Chloromphenicol (Antibiotic) for Agar Plates: 165 ul of chloromphenicol was added to 500 ml of the LB media. This was then poured into circle plates and the agar/lb media was allowed to cool. After plates cooled, condensation was present so plates were left on the bench overnight. How we determined the amount of Kanamycin/Chloromphenicol that would be added to the 500 ml LB media: Antibiotics were added after autoclaving the media. Use the equation C1V1= C2V2 V1= 500 ml LB media C1= 50 ug/ml (How much kanamycin we want) V2= Figuring this out!! C2= 50,000 ug/ml OR 5 mg/ml After doing the calculation, V2= 0.5 ml kanamycin. The working concentration for chloromphenicol is 33ug/mL. Kan 50 ug/ml CAM 25 ug/ml Correct Way C1 is the starting stock solution V1 is the volume that we want to take from the stock solution and put in the media C2 is the Final concentration of antibiotics (usually 1:1000 of the stock solution concentration) V2 is the final volume of the media

2 Making Chemically Competent Cells Friday, May 26, :44 AM 1. 40uL of competent cells. Do not assume the aliquots of cells we made are competent. Transfer the cells to new centrifuge tube to make sure they are competent. 2. Add 5uL of plasmid. Or 1 ul of plasmid depending on what you are working with. 3. Then add 125uL Calcium Chloride and 85uL of H20 4. Total volume should be 250uL 5. Let sit on ice for 30 mins Transformation process: 6. Heat shock at 42 o C for 1 minute 7. Place on ice for 5 minutes. 8. Add 1 ml SOC recovery broth 9. Transfer solution to 1.7mL tube 10. After all solutions have been made, place them in a flask and put on shaker in room for 1 hour at 250 rpm 11. To get more cells, spin the cells at 3000xg for 3 min. Remove most of the supernatant and resuspend the cells

3 Making Competent Cells Friday, August 11, :11 PM Comp cells procedure Day 1 1. Make overnight culture of 5 ml SOB uL DH5a or BL21 cells shaking 37oC SOB a. Use comp cells from first batch of cells we made 40-50uL cells Day 2 1. Check OD 2. Inoculate ~250 ml SOB w/ appropriate volume of overnight culture a. (100x dilution- OD ) (adding ml of culture) b. Dilute w/ SOB and use SOB to blank the spec before using 3. Incubate at 37oC for 1-3 hours until reaches OD 0.3, shaking 4. Spin, resuspend w/ buffer, let sit on ice for 30 min (point of ice is to trick cells into acting as though they are still growing and not give them time to respond to their new environment) 5. Spin, add buffer so OD reaches 1.5, (likely ~50 ml)--- DO 20 ml so that we end up with roughly 100 tubes 6. Aliquot and freeze (200 ul in each tube)

4 DNA Precipitation Protocol 2017 年 7 月 20 日 16:40 DNA Precipitation is a method to increase the concentration of DNA by precipitating it with ethanol. Calculation: 1. For example, you have x ul DNA. 2. x/9 ul of 3M NaOAc should be added *10x/9 ethanol should be added. For example, if you have 100 ul DNA, ul 3M NaOAc should be added. 278 ul ethanol should be added. Then mix, label accurately, and incubate in 20C overnight. The next day, Spin at max speed for 30 min. For clearer instruction on how to place the eppendorf tube into the centrifuge, see hardcopy lab notebook. Decant supernatant liquid, being careful not to disturb the pellet. (Use 200ul pipette to remove). Add 200ul 70% ethanol, spin at max speed for 1 min, then remove supernatant. Air dry for 30 min Add 10 ul of EB buffer (from the QIAgen kit) Vortex ( to make sure that the DNA is mixed well into the eb and the remove the necessity to pipette vigorously as to not damage the DNA) Then obtain concentration DISCLAIMER: KEEP ALL SUPERNATANT LIQUID.

5 Electroporation: Transforming Cells Thursday, May 25, :29 AM How to transform cells using electroporation 1. Add 2uL of plasmid to competent cell solution (which should be anywhere from 50uL to 100uL) and mix 2. Immediately transfer all of the solution (approximately 100uL) to electrode vial 3. Put vial in machine and slide in all the way, press "bacteria" setting, and press "pulse" (1.8kV and 3ms are good numbers) 4. Add 1000uL(1mL) of soc solution(recovery solution) to the vial and mix 5. Transfer solution to 1.7mL tube 6. After all solutions have been made, place them in a flask and put on shaker in room for 1 hour at 250 rpm

6 Gibson Assembly Protocol Monday, July 24, :56 PM This is from page 11 of Products/0AA961B294E444AFBEDD5C4A904C76E6/Datacards%20or%20Manuals/ManualE2611.pdf

7 Gel Extraction Monday, June 5, :17 PM 1. Excise the DNA fragment from the agarose gel with a clean, sharp razor blade. 2. Weigh the gel slice in a colorless tube. Add 3 volumes Solubilizing Buffer (Buffer QG from kit) to 1 volume gel (100 mg gel ~ 100 μl). 3. Incubate on 50 o C water bath for 10 minutes or until gel is completely dissolved in buffer (option to vortex every 2-3 minutes to mix) 4. Add 1 gel volume of isopropanol to sample and mix. 5. Pipet 800 ul of the gel/buffer mixture into a spin column, centrifuge for rpm, discard the flow through from the collection tube, repeat until all of the liquid has been spun down 6. (Optional) Pipet 500 ul solubilizing buffer to the white membrane of the spin column, centrifuge 1min 7. Add 750 ul Buffer PE to the white membrane of the spin column, let sit for 2-5 minutes 8. Centrifuge for rpm, discard flow through 9. Centrifuge for rpm, discard flow through 10. Add 50 ul EB buffer to spin column in clean Eppendorf tube, let stand for 1 min, then spin down for 1min More detail:

8 Inventory Wednesday, May 24, 2017 Glassware 10:33 AM Liquid Reagents/Solvents Solid Reagents Item 500 ml Erlenmeyer flask Quantity 1000 ml bottle ml flask ml bottle 2 Small square bottle 1 medium bottle ml bottle ml bottle 3 25 ml graduated cylinder Test tubes 50 funnel 1 Scalpel 1 Metal spatula 1 small vial ml beaker Dimethyl Sulfoxide BCA protein assay reagent A P1 buffer QG buffer Gold (III) chloride trihydrate p-coumadin acid HEPES D16-1 Dextrose D-Glucose anhydrous Glycerol L-glutamine acid L Histidine L Valine Glycine powder Adenine hemisulfate salt MRS broth powder Imidazole L tyrosine L threonine Ammonium sulfate L serine Uracil L phenylalanine Pentadecane L methionine L tryptophan L lysine Theobromine L arginine Agarose Choline chloride Yeast nitrogen base Tris L aspartic acid LB HiVeg broth modified Calcium chloride dihydrate Bacto peptone Caffeine Luria Broth Agarose grey bottle SDS Bacto Yeast Extract Sodium Chloride Bacto Tryptone

9 Ligase Protocol Wednesday, June 7, :55 AM 20uL Total 2uL Buffer Add 12uL PET28 Add 5uL Cj blue Add 1uL DNA Ligase LAST!!!!! PET28 volume should be approximately double the cj blue volume

10 How to Get Max. Plasmid DNA Tuesday, June 6, :18 PM ml of total cell culture is used. This can be separated in 4 separate falcon tubes at 5 ml each. 2. Centrifuge each falcon tube to form a pellet. 3. Discard supernatant and follow miniprep protocol until it is time to transfer the microfuge tubes to spin column's. 4. Add 2 microfuge tubes worth of supernatant to one spin column. 5. Now have 2 spin columns. 6. Wash as described in normal protocol 7. When eluting, use 75 ul EB and only place on one spin column. 8. Do not discard the elution from one spin column. Instead, use it to elute DNA from the second spin column. 9. Spin again for 30s at max rpm. 10. There should now be 75ul of concentrated DNA in the eluent of the second spin column.

11 Miniprep Protocol Friday, June 2, :51 AM 1. Centrifuge 5-8mL worth of overnight culture 2. Transfer 1.5 ml of bacteria from overnight culture into eppendorf/centrifuge tube 3. Centrifuge for 1 max rpm 4. Add a. 250 ul P1 buffer, pipet up and down to mix b. 250 ul P2 buffer, gently invert to mix c. 350 ul N3 buffer, gently invert to mix 5. Centrifuge 10 13,000 rpm, resulting pellet should be extraneous proteins/rna 6. Transfer DNA supernatant from eppendorf tube to spin column in 800uL increments 7. Centrifuge at top speed for 30 seconds, discard flow through 8. Add 750 ul (.75 ml) of PE buffer 9. Centrifuge again for seconds and discard flow through (at max speed) 10. Centrifuge again for 1 min to remove any residual ethanol 11. Discard collection tube and transfer blue spin column to eppendorf tube 12. Add 50 ul EB directly to center of white membrane of spin column 13. Let stand for 1 min, Centrifuge for 1 min (13,000 rpm) 14. Discard column 15. Use contents of eppendorf tube to measure DNA concentration (ideally 100ng/uL w/ ratio greater than 1.8) Digest 1. Add a. Sterile water b. DNA c. 5uL of 10x Buffer d. 1uL of each enzyme. Always add enzymes last. Example calculation of amount of sterile water and DNA needed: (500ng DNA) / (30 ng/ul) = 16.67uL DNA 43uL uL = 23.6uL water

12 PCR Protocol Wednesday, July 5, :06 PM PCR Protocol for YCP (720 base pairs) and JOE (2300 base pairs) Concept: 1. Melt: Long melt at high temperature to separate double stranded DNA. 2. Melt: Short melt to separate dsdna 3. Anneal: Annealing primers to the DNA for amplification (Should be 5C below primer melting temperature) 4. Extend: Polymerase extends from the primer (Time for reaction is based on length of amplicon). REPEAT STEP times 5. Extend Long extension to finish extending 6. Hold: PCR process is done, hold in temperature (4C) For 50 ul reaction, put these materials into a PCR tube ul 2x Q5 Master Mix ul Primer F ul Primer R 4. 1 ul Template ul ddh2o Normal PCR Reactions JOE 95C 3 min 95C 10 sec 53C 20 sec 72C 1min 15 sec 72C 3 min 12C (or 4C) hold For 72C, do 30 sec / 1 kb YCP 95C 3 min sec 60C 20 sec 72C 45 sec 72C 3 min 12C (or 4C) - hold Settings for the temperature and time are available in the thermocycler under igem file, JOE file and YCP file.

13 Using the Plate Reader Thursday, June 8, :33 PM 1. Spray ethanol onto kim wipe and clean both left and right sides of the plate reader. 2. Use second kim wipe to dry ethanol. 3. Blow off dust with dust blower. 4. Fill one of the wells in the clear rubber container with distilled water. 5. Pipette 2ul of H2O into A1 and A2. 6. Blow off excess dust next to the samples. 7. Place plate reader into loading tray. 8. In the Tecan i-control software: Click on "applications" in the bottom left -> Press "quantify nucleic acid" in the top left-> Ensure that individual blanking box is unchecked-> Highlight A1 and A2 so that they become white -> Click "start blanking" 9. Take out plate reader and wipe down A1 and A2 with kim wipe. Blow off excess dust. 10. Load 2ul of each of your samples into slots of your choosing (usually B1 and B2). 11. Place plate reader into loading tray and highlight the sample slots you want to be measured (so B1 and B2). They should now appear yellow. 12. Click on "start" at the top of the screen. 13. Choose "no" to starting a new measurement. 14. Results should now appear in a new excel file. 15. Make sure to wipe down samples with clean kim wipe and place plate reader back when finished.

14 Plating Transformed Bacteria Thursday, May 25, :25 AM Spread-plating E. Coli Transformants (after lunch) 1. Remove Tubes from the 37 o C shaker after the 1 hour incubation period 2. Label 2 plates for each E. Coli strain/plasmid combo on agar side of plate (include name of strain, date and plate #). Make sure plates are at room temperature and are NOT cold. 3. Pour ~10 plastic balls onto each plate 4. Pipet 200 ul of each sample onto one of their respective plates 5. Centrifuge the remaining 900 ul of each sample ( rpm)- make sure the samples are placed so the centrifuge is balanced 6. Discard the supernatant (extra fluid) into a liquid waste container 7. Pipet up and down to resuspend the pellet in each sample 8. Pipet the remaining liquid (~ 100uL) onto #2 labeled plates

15 Restriction Digest Tuesday, June 6, :54 AM Restriction Digest 1. Add DNA, water, buffer and enzymes to fresh eppendorf tube, making sure to add enzymes last a. DNA volume: (500 ng/ul)/ Measured conc. of DNA b. Water: 43 ul- Volume of DNA calculated c. 5 ul buffer d. 1 ul of each enzyme 2. Vortex for 2 seconds 3. Centrifuge for 10 seconds 4. Let sit on hot water bath 37 o C for hours High Efficiency Restriction Digest Tyler's protocol for efficient restriction digest: 5. Add DNA, water, buffer and enzymes to fresh eppendorf tube, making sure to add enzymes last a. 40 ul of DNA b. 5 ul buffer c. 2.5 ul of each enzyme 6. Let sit on hot water bath 37 o C for 1 hour 7.

16 Making SOC Media Tuesday, August 1, :11 PM For 1000ml (1 liter) volume media: 1. Add the following to 900 ml distilled H2O 20 g bacto tryptone 5 g bacto yeast extract 2 ml of 5M NaCl 2.5 ml of 1M KCl 10 ml of 1M MgCl2 10 ml of 1M MgSO4 20 ml of 1M glucose 1. Add water until 1000 ml total volume 2. Autoclave.