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37 °C

Experiments & Protocols

Protocols

(Temporary edit made by GMC)

1. Colony PCR

Pick a single colony into 5ul of NFW. (Fresh colonies grown that day work best, but they can also come from 4 C).
Boil for 5 min at 95C.

PCR Reaction

Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4 C.

2. Compotent Cell Preparation

For E.coli(DH-5α)

1-) Streak DH5α directly from a frozen stock onto LB agar plate (Amp-).

2-) Incubate at 37℃ for 12 h.

3-) Inoculate one well isolated colony into 5ml of LB(Amp-).

4-) Incubate at 37℃ until OD₆₀₀ =0.8

5-) Transfer the preculture 5ml to 250ml of LB(Amp-)

6-) Incubate at 23℃ until OD₆₀₀ =0.4-0.5 (120rpm/min )

7-) Place sample on ice for 10min

8-) Transfer the culture to steril ice cold tube(250ml)

9-) 3.5Krpm at 4℃ for 5min

10-) Remove the sup well

11-) Resuspend the pellet by gently vortexing and pippeting in 100 ml of ice cold TB*

12-) Sit on ice for 10min

13-) 3K rpm at 4℃for 5min

14-) Remove the sup well

15-) Resuspend the pellet gently in 25ml of TB

16-) Add 875μl DMSO with disposable pippet

17-) Mix gently by swirling

18-) Sit on ice for 10min

19-) Add 875μl DMSO

20-) Mix gently by swirling

21-) Sit on ice for a few minuites

22-) Dispense the sample into 1.5ml tube sterilized with UV for 10min

23-) Chilled in liquid N2

[Preparation TB]

1-) Add the following into 475 ml milli-Q

2-) PIPES 1.5g

3-) CaCl2･2H2O 1.1g

4-) KCl 9.3g

5-) Adjust pH to 6.7 with 1N KOH

6-) Add 5.45g of MnCl2･4H2O

7-) Add milli-Q upto 500ml

8-) Filtration

3. Gel Electrophorosesis

Standard %0.8 Agarose Gel Preparation

Measure out 0.8 g of agarose
Pour agarose powder into a microwavable flask along with 100mL of 1xTBE
Microwave for 3 mins (until the agarose has dissolved completely and there is a nice rolling boil).
Let agarose solution cool down for 5min.
Add 3.6 ul EtBr and pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).

Wait 20-40 mins until poured gel has completely solidified.

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4.Gel Purification

Gel Purification

Gel purification using&Thermo Scientific GeneJET Gel Extraction Kit

-All purification steps should be carried out at room temperature.

-All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g

Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.

Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).
Note. For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice.
Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
Optional: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly.
Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.
Optional: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Add 700 ul of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.
Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of NFW to the center of the purification column membrane. Centrifuge for 1 min.
Note. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65°C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation.
Discard the GeneJET purification column and store the purified DNA at -20°C.

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5. LB Agar Preparation

1-) Add 200 mL of dH2O to a graduated cyclindar.

2-) Transfer dH2O into glass bottle.

3-) Add 7 gr of LB-agar powder

4-) Autoclave the bottle.

5-) After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)

6-) Pour the plates .

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6. LB Broth Preparation

1-) Add 200 mL of dH2O to a graduated cyclindar.

2-) Transfer dH2O into glass bottle.

3-) Add 4 gr of LB powder

4-) Autoclave the bottle.

5-) After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)

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7. Midiprep

Midiprep Using invitrogen Midiprep Kit

Preparing Cell Lysate

1. For high copy number plasmids, use 15–25 mL of an overnight LB culture per sample in a disposable 50-mL conical tube.

Note: If you are using >25 mL of culture volume of high copy plasmids, add twice the amount of Resuspension Buffer (R3) with RNase A, Lysis Buffer (L7), and Precipitation Buffer (N3) as directed in steps 3, 4, and 5, below, for best results.

For low copy number plasmids, use 25–100 mL of an overnight LB culture per sample in a 50-mL tube.

2. Harvest the cells by centrifuging the overnight LB culture at 4000 × g for 10 minutes. Remove all medium.

3. Add 4 mL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the cells until homogeneous.

4. Add 4 mL Lysis Buffer (L7). Mix gently by inverting the capped tube until the lysate mixture is thoroughly homogenous. Do not vortex. Incubate at room temperature for 5 minutes.

Note: Do not allow lysis to proceed for more than 5 minutes.

5. Add 4 mL Precipitation Buffer (N3) and mix immediately by inverting the capped tube until the mixture is thoroughly homogeneous. Do not vortex.

6. Centrifuge the mixture at >12,000 × g for 10 minutes at room temperature.

Note: If the pellet does not adhere to the bottom of the tube, incubate the tube at room temperature for 5 minutes to allow the lysate and gelatinous pellet to separate. Pipet the clear lysate into another, sterile tube and centrifuge at >12,000 × g at room temperature for 5 minutes to remove any remaining cellular debris.

7. Proceed to Binding and Washing DNA, next page.

Binding and Washing DNA

1. Load the supernatant from step 6 (Preparing Cell Lysate) onto the equilibrated column. Allow the solution in the column to drain by gravity flow.

2. Wash the column twice with 10 mL Wash Buffer (W8). Allow the solution in the column to drain by gravity flow after each wash. Discard the flow-through.

3. Proceed to Eluting and Precipitating DNA. For DNA precipitation, you can use the PureLink® HiPure Precipitator Module (page 34) which allows you to precipitate DNA within 10 minutes without using a centrifuge. Alternatively, follow Eluting and Precipitating DNA on page 14 to perform traditional DNA precipitation using centrifugation. Refer to the manual supplied with the PureLink® HiPure Precipitator Module for a detailed protocol.

Eluting and Precipitating DNA

1. Place a sterile 15-mL centrifuge tube (elution tube) under the column.

2. Add 5 mL Elution Buffer (E4) to the column to elute the DNA. Allow the solution to drain by gravity flow. Do not force out any remaining solution. The elution tube contains the purified DNA. Discard the column.

3. Add 3.5 mL isopropanol to the elution tube. Mix well.

Note: Proceed to the protocol described in the PureLink® HiPure Precipitator manual after this step, if you are using the precipitator module.

4. Centrifuge the tube at >12,000 × g for 30 minutes at 4°C. Carefully remove and discard the supernatant.

5. Resuspend the pellet in 3 mL 70% ethanol.

6. Centrifuge the tube at >12,000 × g for 5 minutes at 4°C. Carefully remove and discard the supernatant.

7. Air-dry the pellet for 10 minutes.

8. Resuspend the DNA pellet in 200 µL TE Buffer (TE). For low copy number plasmids, use 100 µL of TE Buffer.

Note: Occasionally, insoluble particles may be present. These particles do not influence the quality of the DNA and can be easily removed. To remove insoluble particles, centrifuge the DNA solution at high speed for 1 minute at room temperature. Transfer the supernatant (DNA sample) into a fresh tube. Storing DNA To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at –20°C for long-term storage.

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8. Miniprep

Miniprep

Miniprep using Thermo Scientific GeneJET Plasmid Miniprep Kit

-All purification steps should be carried out at room temperature.

-All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g

Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
Note. Ensure RNase A has been added to the Resuspension Solution.
Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note.Do not add bleach to the flow-through.
Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
Repeat the wash procedure (step 7) using 500 ul of the Wash Solution.
Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the NFW to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min. Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids sup20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
Discard the column and store the purified plasmid DNA at -20°C.