Team:Wisconsin-Madison/protocols

Plasmid DNA extraction

Alkaline Lysis

Alkaline Lysis is for screening of plasmids

1. Pellet the overnight culture(s) in a 1.5 ml or 2ml eppendorf tube. (I usually do 10,000 rpm, 3 minutes) 1 minute works fine. I usually use 3 ml culture per prep.
2. Resuspend each pellet in 200 μl Alkaline Lysis Sol I, RnaseA added (final RNase A concentration should be 100 μg/mL). Make sure there are no lumps.
3. Add 400 μl Alkaline Lysis Sol II. Invert 4-6 times to mix. Do not allow reaction to lyse for more than 5 min. Sample should clarify.
4. Add 300 μl Alkaline Lysis Sol III. Invert 4-6 times to mix. Sample should have a white precipitate.
5. Add 100 μl chloroform. Do this in a fume hood. Invert 4-6 times to mix.
6. Rest on ice for 5-10 minutes. This step is so that the chloroform does not get too hot in the centrifuges and leak out of the tubes. If you want to skip this step you might consider using less chloroform. I put the tubes at -20 for a couple of minutes.
7. Centrifuge at max. speed (14,000 rpm) for 10 minutes.
8. Pipet 750μl of supernatant/aqueous layer into a fresh tube. I do up to 800 uL
9. Add 1/10 volume (75μl) 3M NaOAc, pH 5.2. Vortex/flick to mix. 80 uL
10. Add 0.7-1.0 Volume COLD isopropanol. Vortex/flick to mix. If in a hurry go straight to step 11, otherwise rest on ice for 10-30 minutes. I have even let it precipitate overnight at 4°C if convenient. 600 uL isopropanol. Then I put it at -20 for 5 minutes up to over the weekend if needed.
11. Centrifuge at max. speed for 25 min. Most miniprep protocols say to do this at 4°C, but I have not noticed decreased yield by centrifuging at room temp.
12. Remove and discard the supernatant. Don’t disturb the pellet. Sometimes I can’t see a pellet, and more often than not I still have DNA.
13. Add 1ml of 70% EtOH (at room temp.). Invert 4-6 times to rinse the tube.
14. Centrifuge at max speed for ~5 minutes. Room temp. is fine. Remove and discard the EtOH.
15. Repeat steps 14 and 15 to remove all traces of isopropanol. Pulse spin after removing bulk of final EtOH wash and pipet off remaining EtOH.
16. Air dry the pellet for ~15 minutes (pellet will change from white to clear as it dries). Resuspend in desired volume of H2O or T10E1, depending on downstream applications. If you pipet off the EtOH well, then I have done this for as little as 2 minutes before. For fosmids, I usually resuspend the pellet in 20 uL water.

Kit

Use the kit when you need very clean DNA. Ex cloning, sequencing

1. Refer to kit instructions

Digestion

Screening

For screening, you only need a small amount to run on a gel - 10uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

• DNA - 2uL (usually fine)
• Buffer(10x) - 1uL
• BSA(10x) - 1uL
• Enzyme - 0.4uL each (ADD LAST and no more than 10% of rxn volume)
• Water - fill to 10uL

1-2 hours in 37C waterbath (check NEB if you want quicker time)

• Add 2uL of 6x Dye
• Load 6uL in gel

Cloning

During cloning, you will need to digest more DNA for gel extraction - 50uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

• DNA - 2ug
• Buffer(10x) - 5uL
• BSA(100x) - 0.5uL
• Enzyme - 2uL each (ADD LAST and no more than 10% of rxn volume)
• Water - fill to 50uL

1-2 hours in 37C waterbath (check NEB if you want quicker time)

• Add 10uL of 6x Dye
• Load 60uL in gel

Template Destruction

If your product for digestion came directly from PCR you can destroy the original template by preforming a DpnI digestion. DpnI will digest methylated DNA. PCR product is unmethylated. If needed, do this step before cloning digestion.

• 1uL DpnI/50uL rxn
• incubate in 37C waterbath for 1 hour

Gel Extraction

Use the kit and refer to kit instructions

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose."
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl). For example, add 300 μl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix. For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube. Collection tubes are re-used to reduce plastic waste.
9. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after addition of Buffer PE, before centrifuging.
10. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm). IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alter- natively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

Ligation

1. measure the concentration of the inserts and the vectors.
2. use In-Fusion® Molar Ratio Calculator from Clontech to calculate the mixing ratio of the inserts and vectors. Insert/Vector Ratio: 3-5
3. once the amount of inserts and vectors has been calculated, make a calculation for a 10ul total volume reaction
4. After the calculation has been done, get a small tube and label it
5. place the T4-buffer on ice to let it dissolve
6. Add the insert and vector into the tube, mix (Add water to compensate if needed)
7. Add 1ul T4-buffer into the mixing solution
8. Add 1ul Ligase into the mixing solution
9. Either leave it on the bench for a bench-top ligation for 1-2 hours OR put into the thermocycle at 16C for overnight

Transformation

1. Clean the cuvette and UV for 15mins.
2. Locate Electroporator source and cuvette holder.
3. Thaw required number of frozen cell aliquots on ice
4. Thaw required DNA on ice
5. Place the clean cuvette on ice
6. mix 0.5ul to 1.0ul DNA with 40ul competent cells
7. let it sit on ice for 2-5mins.
8. transfer the DNA-Cell mixture into the cuvette
9. Place the cuvette in the holder
10. Have 960ml of LB broth ready
11. Press the button to "shock" the cells
12. Immediately put 960ml LB into the cuvette and mix well
13. Transfer the cells into a 1.5ml centrifuge tube
14. Place tubes in 37C shaker 1-1.5hour.

Plating

1. Place the plate with the correct antibiotic in the 37C incubator 1 hour before the plating for pre-warming
2. Get the transformed cells and centrifuge at 2500rpm for 5 mins.
3. Discard about 850ul of supernatant, and re suspend the rest of the cells.
4. Put the liquid culture (about 150ul) onto the LB agar plate
5. Use a glass sticks to spread out the cells
6. Place the plate in the 37C incubator with the bottom facing upward

PCR clean-up

Use the kit and refer to the kit instructions as follows

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
4. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.
5. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
6. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min at maximum speed. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
7. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer. (Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.)

Colony PCR

1. Lyse a single colony in 50uL water
2. Vortex
3. Plate 2uL
4. Use 5uL for DNA template (see components below)

For 10uL total Rxn

• 2.9 uL water
• 5 uL 5x GoTaq Master Mix
• 0.05uL Forward Primer
• 0.05uL Reverse Primer
• 2uL DNA Template

Electro-competent Cells

The process consists of growing cells to mid-log stage, harvesting, and performing multiple washes with sterile 10% glycerol to remove salts which interfere with electroporation.

General Considerations:

• Keep everything cold, on ice
• Glycerol pellets are not firm; try to remove as much supernate as possible, but be careful not to lose the pellet
• All containers that come in contact with cells should be sterile
• Keep centrifuge bottles dedicated for making Electrocompetent cells
• Have 1 liter of 10% sterile glycerol chilled on ice, to less than 4C... or in a cold box overnight.
• Keep manipulation of cells to a minimum, be gentle.
• Resuspend pelleted cells using a sterile plastic pipette. Work quickly.
• Harvest cells at 0.6 – 0.75 O.D. (A600nm)

Fermentation:

• Inoculum:
  • Streak for single colony from -70C glycerol stock
  • Start 50 ml, No Salt LB inoculum, 37C, overnight
• Fermentation
  • Use 25 ml of the above Inoculum per liter of No Salt LB media (prewarm media to 37C)
  • Grow at 37C, shake at approximately 200 rpm
  • Grow to 0.6 – 0.75 O.D. (A600nm)......transfer to ice immediately to chill

Processing

1. Spin the chilled culture at 8,000 rpm, 10 minutes, 2 degrees C (use four 250 ml centrifuge bottles). Remove the supernate carefully. Save the pellets.
2. Resuspend all four pellets in a total volume of 200 ml cold 10% glycerol. Combine all resuspended pellets in one 250 ml centrifuge bottle.
3. Spin at 8,000 rpm, 10 minutes. Remove the supernate carefully.
4. Resuspend pellet in 150 ml cold 10% glycerol.
5. Spin at 8,000 rpm, 10 minutes. Remove the supernate carefully.
6. Resuspend pellet in 100 ml cold 10% glycerol.
7. Spin at 8,000 rpm, 10 minutes. Remove the supernate carefully.
8. To the pellet, add 2 ml 10% glycerol. Resuspend carefully with a 1 ml Pipetteman.
9. Transfer 110 ul of resuspended cells into cold***(-70C) 1.5 ml microcentrifuge tubes.
10. Transfer immediately to a -70C freezer (Do not use liquid nitrogen).
11. Freeze overnight before using cells.

Freeze Stock

Mix in cryotube. Make three stocks for each sample; place one in the working box and two in the backup box. Document placement of samples of lab site.

• 750uL overnight culture
• 250uL sterile 60% glycerol

Diluting Primers

Dilute primers to 60pM

Using the Autoclave

Check water level. Add water until the water level almost reaches the lower compartment. Close the exhaust.

• Time: 15 minutes
• Temperature: 121C
• Start

Plate Reader Protocol

Preparing Cultures:

1. Make a 10 mL overnight culture of the specific organism to be tested.
2. Measure the OD of the overnight culture in the morning.
3. Dilute the culture to 0.01 OD with LB.
4. Put the 0.01 OD culture in the incubator at 37oC and 250 rpm and grow to 0.05 OD. This takes approximately 2 hours.
5. Measure the OD of the culture and record the volume of culture removed to ensure appropriate inducers concentrations at later stages.
6. If the OD is at 0.05, add the correct concentration of inducer to the volume of culture (for a pBAD system, add 0.2% arabinose, for a Plac system, add 1mM IPTG). (If the OD is below 0.05, continue incubating the culture and repeat steps 5 and 6 at a later time. If the OD is above 0.05, dilute with LB to 0.05 OD and add all appropriate antibiotics and reagents to maintain desired concentration levels. Add the corresponding amount of desired inducer.)
7. To the pellet, add 2 ml 10% glycerol. Resuspend carefully with a 1 ml Pipetteman.
8. Transfer 110 ul of resuspended cells into cold***(-70C) 1.5 ml microcentrifuge tubes.
9. Transfer immediately to a -70C freezer (Do not use liquid nitrogen).

Plating Cultures:

1. UV sterilize the plate and lid for 15 minutes under a UV hood.
2. Each well can contain 200 µL , so to produce triplicate results of a desired concentration, add the corresponding amount of culture down a column for three rows. (IE. For example, add 199 µL culture to produce a 0.5% concentration of analyte down a column.)
3. To produce a concentration gradient along a row, decrease the amount of culture added and repeat down the same column to produce a triplicate result. (IE. For example, add 200 µL, 199 µL, 198 µL of culture along a row if a 0%, .5%, 1% concentration gradient of analyte is desired.)
4. Down each column, add the corresponding amount of analyte to the wells containing culture. (For example, add 1 µL of analyte to a well containing 199 µL culture if a concentration of 0.5% analyte is desired. )

Reading the Plate: Method A.

1. Once the plating of culture and analyte is complete, cover the plate with the lid and secure into an incubator at 37oC and 250 rpm overnight.
2. Remove the plate from the incubator and place in the plate reader.
3. Set the excitation and emission wavelengths to correspond to the fluorescent protein being used.
4. Set the plate reader to record OD.
5. Run the plate reader.
6. Divide each of the fluorescence readings by the OD to normalize the results.
7. Produce a plot of normalized fluorescence versus the concentration gradient of analyte in question.

Reading the Plate: Method B.

1. Place the plate directly in the plate reader set at 37oC and 250 rpm and record the fluorescence and OD on a time course.
2. After your desired period of time, normalize the fluorescence by dividing the fluorescence by the OD.
3. Produce plots of the normalized fluorescence at different concentrations through time.