Team:Lubbock TTU/Experiments
Protocols
LB Media:250 ml d H20
5 g LB lenox media
1) Pour 250 ml of water into a plastic beaker with a magnetic spin vain.
2) Measure 5 g of LB lennox media on a weigh boat.
3) Add the LB lennox media slowly to the stirring water.
4) After the solution is homogeneous, pour 250 ml into a glass bottle.
5) Make sure the glass bottle is label and has autoclave tape.
6) Autoclave = liquid 30. LB Agar:
250 ml d H20
5 g LB lenox media
3.75 g Agar
1) Pour 250 ml of water into a plastic beaker with a magnetic spin vain.
2) Measure 5 g of LB lennox media on a weigh boat.
3) Add the LB lennox media slowly to the stirring water.
4) Weight 3.75 g of Agar and pour it directly into a 500 ml glass bottle.
5) After the LB lennox solution is homogeneous, pour 250 ml into the glass bottle.
6) Make sure the glass bottle is label and has autoclave tape.
7) Autoclave = liquid 30. LB Liquid Cultures:
1)Take 2 culture tubes with the lids on and place them in a glass beaker.
2)Microwave for 5 minutes at power level 4.
3)After the microwave is finished take the tubes into the clean bench.
4)Wipe the clean bench down with ethanol before and after use.
5)Label the tubes appropriately.
6)Add 5 ml of LB media to each tube and then add appropriate antibiotic.
7)Flame a loop and pick a colony from a petri dish.
8)Grow overnight at 28-30 °C. PLATES:
1)250 ml LB Agar and microwave for 1 minutes, and then 30-second intervals until is completely melted. Do not let it boil. let the Agar cold down to the point it only feels warm to the touch.
2)obtain two 50 mL conical vials.
3)after the agar is cold down enough pour it into one 50 ml into the conical vial.
4)dd appropriate concentration of antibiotic.
5)label plates. MINIPREP:
This protocol was obtain from QIAprep Miniprep Handbook
QIAprep Spin Miniprep Kit using a Micro centrifuge
1) Make 1-5 ml overnight cultures of E.coli in LB media.
2) Pelleted Bacterial cell by centrifuging at 45000 g for 10 minutes.
3) Discard supernatant into a container.
4) Resuspend pelleted bacterial cell in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
5) Add 250 µl Buffer P2 and invert the tube 4-6. The lysis reaction should not proceed for more than 5 minutes
6) Add 350 µl Buffer N3 and invert 4-6 times. (Solution should be cloudy)
7) Centrifuge for 10 minutes at 13,000 rmp.
8) Obtain the supernantants from step 4 and add 800 µl to a spin column by pipetting.
9) Centrifuge for 1 minutes and discard flow- through.
10) Wash spin column by adding 750 µl of Buffer PE and centrifuge for 1 minute. Discard Flow-through
11) Transfer spin column to a clean 1.5 ml Eppendorf tube and centrifuge for 1 minute.
12) Transfer spin column to a new clean 1.5 ml and elute DNA by adding 50 µl of water. (you can warm up the water to improve the efficient
13) Let it stand for 1 minutes and centrifuge for 1 minute,
14) Obtain the DNA concentration.
file:///C:/Users/pgaravit/Downloads/E2.QIAprep.Miniprep.Handbook.pdf Electrocompetent Protocol:
This is according to the Molecular Cloning (Sambrook) book, page 178 book 1.
Day 1 - Make a 5mL starter culture:
Day 2 - Make a 50 mL cell culture:
1)Measure 1 g of Agarose and 100 ml of 1x TAE buffer in specific flask designated for ethidium bromide
2)Weigh the total of flask and mixture
3)Microwave mixture for 1 minute and power level 10
4)Stir and microwave once more for one minute at power level 10
1)Since water has evaporated, weigh the mixture and flask again and add enough water to compensate the amount that evaporated in the microwave
2)Set up the casting apparatus
1)Add 4 µl ethidium bromide to the mixture and swirl bottle to homogenize
2)Pour mixture into the apparatus. Pop the bubbles with a pipette tip. Keep the bubbles away from the wells
3)Let the gel solidify for 15-20 minutes (should turn opaque when ready)
4)Since you can only add 10 µl into the well:
1)Remove comb carefully from gel and apparatus
2)Fill the apparatus with the 1x TAE buffer so that the gel is completely covered (If it's already full then don't fill it up with new buffer.)
3)Grab the correct DNA ladder and pipette 10 µl into the far left lane
4)Load 10 µl of each sample from the parafilm wells
5)Turn on the machine at 110 V for 20-30 minutes and make sure bubbles form PFU PCR:
Recipe:
Colony PCR: 1) Use an autoclave toothpick to pick a colony and place the toothpick tip into a PCR tube at the very bottom.
2) Make a Master Mix of the following
§ 0.5 µl FWD primer
§ 0.5 µl REV primer
§ 4.0 µl of 5X Tag Go buffer
§ 13.5 µl of dWater
§ 1 µl Tag enzyme
3) Add corresponding amount to each PCR tube
Chemical Competent Protocol: 1. Pick a single bacterial Colony from a plate that has been incubated for 16-20 hours at 37 C
2. Transfer colony into 5 ml of LB broth in a culture tube
3. Incubate culture for overnight at 37 °C with vigorous shaking (200 rpm)
1. Inoculate two 250 ml flask containing 50 ml of LB media with 500 ul of your overnight culture
2. Read the OD600 of all three cultures. Continue to monitor every 45 min until the reading is at 0.55
3. Transfer the culture vessel to ice water bath for 10 min
4. Harvest cells by centrifugation at 2500 g for 10 minutes at RT in 50 ml falcon tube
5. Pour off medium and dry the tube (inverted) on paper towels for 2 min
6. Resuspend the cells gently by swirling in 16 ml of ice-cold Inoue transformation buffer to each falcon tube
7. Harvest cells by centrifuge at 2500 g for 10 minutes at room temperature
8. Pour off medium and dry tube inverted on paper towels for 2 min
9. Resuspend cells gently in 4 ml of Inoue transformation buffer to each falcon tube
10. Add 300 µl of DMSO to each falcom tube (swirl to mix bacterial suspension)
11. Store on ice for 10 min
12. Quickly dispense 50 µl aliquots of suspension into chilled, sterile microfuge tubes
13. Snap freeze competent cells in liquid nitrogen ( store stock at -80 °C) Electroporation Protocol:
1) Preheat SOC medium to 42°C or thaw out the SOC medium.
2)Thaw out electrocompetent cells on ice for 5-10 minutes
3)Add the necessary volume (in μL) of DNA and into an electrocompetent cell tube. We used 2-3µL for linear fragments and 1µL for plasmid
4)Incubate on ice for 10 mins
5)Move the cells into 1mm cuvette, which should also be on ice.
6)Electroporate on Ec1.
7)Add 500µL of preheated SOC medium to the 1mm cuvette and transfer to a fresh 1.5mL Eppendorf tube.
8)Incubate tubes at 37°C for 3 hours without shaking
9)Plate 50µL onto plates
10)Incubate overnight at 37°C. Genomic PCR:
1)label colonies you are going to use
2)add 10 µL of water to each PCR tube
3)Pick the colony with a toothpick and mix it into PCR tubes (Keep Track of what plate and colony you are using)
4)boil at 96 °C for 15 min and let them cool down
5)make a master mix while you wait
6)obtain the PCR tube, mixed then pipette mix them one more time
7)get new PCR tubes, add 19 µl of master mix to new PCR first, then add 2 µl from the PCR tubes containing the colony. make sure to only get clear liquid (white stuff is junk)
8)add 1 µl of Tag
Chemical Competent Cell Transformation:1. add 10 µl of water to the specific wells
2. obtain Chemical Competent Cells and SOC media
3. add 1 µL of the corresponding plasmid to cell and wait for two minutes
4. heat shock cells at 42 °C for 45 seconds and then put them on ice for two minutes
5. add 250 µL of SOC media to each tube
6. incubate at 37 °C for an hour
7. proceed to plate the cells ZymoPURE Plasmid Midiprep :
1)ZymoPURE Plasmid MiDiprep
This protocol was obtain from ZymoPURE
Buffer Preparation:
If the ZymPURE P2 and ZymoPURE Binding buffer precipitate incubate the bottless at 30-37 C for 10-20 minutes and mix by inversion (DO NOT MICROWAVE) BEFORE STARTING:
Plasmid Midiprep Protocol (should be performed at room temperature (15-30 C)
1)Add 8 ml of ZymoPURE P1 (RED) To the bacterial cell pellet and resuspend completely by vortezing or pipetting
2)Add 8 ml of ZymoPURE P2 (GREEN) and immediately mix by gently inverting the tube 6 times. (Do not vortex)
3)let it sit at room temperature for 2-3 minutes (cells are completely lysed when the solution appears clear, purple, and viscous
4)Add 8 ml of ZymoPURE P3 (YELLOW) and mix gently but thoroughly by inversion (Do not Vortex)
5)The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form
6)Ensure the plug is attached to the luer lock at the bottom on the ZymoPURE syringe Filter.
7)Place the syringe filter upright in a tube rack and load the lysate into the ZymoPURE Syringe
8)push the solution through the ZymoPURE Syringe filter to clear the debris (Save the cleared lysate)
9)Add 8 ml ZymoPURE Binding Buffer to the cleared lysate from step 5 and mix thoroughly by inverting the capped tube 8 times. Centrifugation Protocol:
1)Remove the 50 ml Reservoir from the top of the Zymo-Spin™ III-P Column Assembly. Ensure the connection between the 15 ml Conical Reservoir and Zymo- Spin™ III-P column is finger-tight and place the assembly into a 50 ml conical tube.
2)Add 10 ml of the mixture from step 6 into the 15 ml Conical Reservoir/ZymoSpin™ III-P Column assembly, and centrifuge at 500 x g for 2 minutes. Empty the 50 ml conical tube and repeat this step until the entire sample has passed through the column.
3) Add 2 ml of ZymoPURE™ Wash 1 to the Zymo-Spin™ III-P column assembly and centrifuge the column at 500 x g for 2 minutes.
4)Add 2 ml of of ZymoPURE™ Wash 2 to the Zymo-Spin™ III-P column assembly and centrifuge the column for 2 minutes at 500 x g. Repeat the wash step.
5)Unscrew the purple Luer Lock cap from the top of the Zymo-Spin™ III-P Column and discard the 15 ml Conical Reservoir. Place the Zymo-Spin™ III-P Column in a Collection Tube and centrifuge at ≥ 10,000 x g for 1 minute, in a microcentrifuge, to remove any residual wash buffer.
6)Transfer the column into a clean 1.5 ml microcentrifuge tube and add 200 µl of ZymoPURE™ Elution Buffer 1,2,3 directly to the column matrix. Wait 2 minutes, and then centrifuge at ≥ 10,000 x g for 1 minute in a microcentrifuge.
7)Optional: For EndoZero Plasmid DNA4 , place the EndoZero™ II Spin-Column in a clean 1.5 ml microcentrifuge tube. Add the entire eluate from step 12 into the EndoZero™ Spin-Column, wait 2 minutes, and then centrifuge at 5,000 x g for 1 minute in a microcentrifuge. Store the eluted plasmid DNA at ≤ -20°C. Restriction Digest of Backbone:
This protocol was obtain from NEBiolabs.
Gather materials. PstI, EcoRI-HF and 10x NEB Buffer 2.1 and concentrated backbone (pSB1C3) from the plasmid box.
Keep all materials, especially enzymes, on ice
Obtain a pcr tube + cap from
1)Add 3.0 µL of NEB buffer 2.1 to each tube.
2)Add 1.0 µL of EcorI-HF to tubes 1-4
3)Add 1.0 µL of PstI to tubes 1-3 and 5
4)Total volumes should be 30µL
5)Mix and spin briefly
6)Incubate at 37 °C for 1 hour
7)Run 2 µL of the sample on a gel against 1µl of the the uncut backbone.
8)If gel is positive, proceed to the next step.
9)Heat kill at 80 °C for 20 minutes
10)Store digest product in the refrigerator Ligation Protocol with T4 DNA Ligase (M0202):
1)This protocol was obtain from NEBiolabs.
1. Gently mix the reaction by pipetting up and down and microfuge briefly.
2. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
3. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
4. Heat inactivate at 65°C for 10 minutes.
5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.