Restriction Digests

Materials

• Ice and bucket/container
• DNA to be digested
• Buffer M (10x)
• Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
• Incubator

Process(an example)

1. Add 47ul of DNA to be digested into a 1.5ml microcentrifuge tube.
2. Add 5.0ul of10x buffer M.
3. Add 1.0ul of EcoRI.
4. Add 1.0ul of SpeI.
5. There should be a total volume of 50ul. Mix well and spin down briefly.
6. Incubate the restriction digest at 38C for 30min. We incubate in an incubator.
7. Run a portion of the digest on a gel (6ul), to check that part length is accurate.

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Ligation

1. Add digested fragment A.
2. Add digested fragment B.
3. Add ligation mixture.
4. Ligate 16C/30 min.

Note: Make the amount of fragment A and B equimolar. And make sure that the volume of ligation mixture is equivalent to the total amount of the volume of fragment A and B. we used a constant temperature water bath.

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Transformation

1. Deal 25ul of competent cells to each 1.5ml tubes.
2. Add 1ul of each solution made by ligation to each tube.
3. Close tubes and incubate the cells for 30 min on ice.
4. Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 seconds.
5. Incubate the cells on ice for 2 minutes.
6. Add 225ul of SOC media to each tube.
7. Incubate cells at 37C for 30 minutes.
8. Inoculate with each solution of cells to each plate with appropriate antibiotics.
9. Culture the cells.

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Densitometry(Nanodrop,In the case of nucleic acid)

1. Activate the device.
2. Choose “Nucleic Acid” at main menu.
3. Wash a space of a sample with distilled water and then wipe gently the top and bottom of the arm.
4. Choose a sample type. There are DNA-50(double-strand), DNA-33(single-strand), RNA.
5. Pour distilled water to do a blank test and then wipe the top and bottom of the arm.
6. Pour the sample to measure the concentration.

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Colony PCR

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Electrophoresis

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Miniprep

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SDS-PAGE

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Gel Extraction with FastGene™ Gel/PCR Extraction Kit

Separation of the gel

1. Cut down a DNA fragment from an agarose gel. Remove surplus agarose to make the gel fragment as small as ossible.
   Note: Recommended concentration of agarose is under 2.5%.
2. Transfer the gel fragment(max 300 mg) to a centrifugal tube.
3. Add 500 µL of buffer for combination GP1 to a sample and then stir it with a vortex mixer.
4. Incubate a sample at 55 C° for 10~15 minutes (until the gel fragment completely dissolves). While incubating invert the tube every 2~3 minutes.

DNA bonding

1. Insert FastGene™GP column into a collection tube.
2. Dispense (max 800 µL of) sample solution of the previous step into FastGene™GP columns and then centrifuge them(13,000 rpm 30 seconds).
3. Throw filtrate away and then return the column to the collection tube.
   Caution: If the volume of the sample solution is over 800 µL, repeat the step of DNA bonding.

Washing

1. Add 600 µL of washing buffer GP2 to a FastGene™GP column and then centrifuge it(13,000 rpm 30 seconds).
2. Throw filtrate away and then return the column to the collection tube.
   Caution: If you use TAE gel, proceed to the next step. If you use TBE gel, repeat the step of Washing to completely remove boric acid.

Desiccation

1. Centrifuge a column matrix(13,000rpm 2 minutes) to desiccate it.

DNA Elution

1. Insert a FastGene™GP column into a new centrifugal tube.
2. Add 20~50 µL of elution buffer GP3 to the center of column matrix.
3. Until elution buffer is absorbed by the column matrix, keep the same state for 2 minutes.
4. Centrifuge it(13,000 rpm 2 minutes) and then elute refined DNA.
   Caution: The yield of a large DNA fragment(more than 5 kbp) can be increased by the use of advance heated(70 C°) buffer.

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PCR Purification with FastGene™ Gel/PCR Extraction Kit

Sample preparation

1. Add buffer for combination GP1 to a sample in the ratio of one to five(e.g. 200 µL of buffer for combination GP1 per 40 µL of PCR reaction liquid).
   Caution: If the volume of the sample is under 40 µL, add buffer for combination GP1 or water for PCR to adjust the volume of the sample to 40 µL.
2. Stir it with a vortex mixer.
3. If the gross of volume of buffer for combination GP1 and water for PCR exceed the capacity of a PCR tube, transfer water for PCR to a centrifugal tube.

DNA bonding

1. Insert FastGene™GP column into a collection tube.
2. Dispense the sample solution of the previous step into FastGene™GP columns and then centrifuge them(13,000 rpm 30 seconds).
3. Throw filtrate away and then return the column to the collection tube.

Washing

1. Add 600 µL of washing buffer GP2 to a FastGene™GP column and then centrifuge it(13,000 rpm 30 seconds).
2. Throw filtrate away and then return the column to the collection tube.

Desiccation

1. Centrifuge a column matrix(13,000rpm 2 minutes) to desiccate it.

DNA Elution

1. Insert a FastGene™GP column into a new centrifugal tube.
2. Add 20~50 µL of elution buffer GP3 to the center of column matrix.
3. Until elution buffer is absorbed by the column matrix, keep the same state for 2 minutes.
4. Centrifuge it(13,000 rpm 2 minutes) and then elute refined DNA.
   Caution: The yield of a large DNA fragment(more than 5 kbp) can be increased by the use of advance heated(70 C°) buffer.

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Prorein Purification with Ni-NTA

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HisLink Spin Protein Purification

Materials to Be Supplied by the User

• Nuclease-Free Water
• rotor
• wide-bore pipette tips
• NaCl or 5M NaCl solution for use with HQ-tagged proteins
• tabletop centrifuge
• 1.5ml microcentrifuge tubes

Centrifugation Protocol

1. Pipet 700μl of bacterial culture into a 1.5ml microcentrifuge tube. Add 70μl of the FastBreak™ Reagent/DNase I solution prepared in Section 3.D, Table 1, to the culture.
   Note: For HQ-tagged proteins, please see Section 3.C.
2. Resuspend the resin and allow it to settle. Once the resin has settled, use a wide-bore pipette tip to transfer 75μl of the HisLink™ Resin from the settled resin bed to the 1.5ml microcentrifuge tube. To successfully transfer resin, place the wide-bore pipette tip deep into the resin and pipet slowly to assure that a consistent amount of resin is drawn into the pipette. Allow the resin to resettle between each pipetting.
   Note: We recommend optimizing the amount of HisLink™ Resin used for low- (<1mg/ml) or high- (>1mg/sample) expressing proteins. For lowexpressing proteins, less resin should be used; similarly, for highexpressing proteins, more resin per sample can be used.
3. Incubate the sample and resin for 30 minutes, mixing frequently on a rotating platform or shaker to optimize binding.
4. Place a Spin Column onto a Collection Tube (or a new 1.5ml microcentrifuge tube). Use a wide-bore pipette tip to transfer the lysate and resin from the original 1.5ml microcentrifuge tube in Step 3 to the spin column. If resin remains in the 1.5ml microcentrifuge tube, add HisLink™ Binding/Wash Buffer to the tube, then transfer the buffer and remaining resin to the spin column.
5. Centrifuge the spin column with the collection tube for 5 seconds or until the liquid clears the spin column.
6. To save the flowthrough, remove the spin column from the collection tube and transfer the flowthrough from the collection tube to a new 1.5ml microcentrifuge tube. Otherwise, discard the flowthrough.
7. Place the spin column back onto the collection tube. Add 500μl of HisLink™ Binding/Wash Buffer to the spin column, then cap the spin column. Centrifuge for 5 seconds or until the Binding/Wash Buffer clears the spin column. Discard the flowthrough. Repeat for a total of two washes.
8. Take the spin column off the collection tube and wipe the base of the spin column with a clean absorbent paper towel to remove any excess HisLink™ Binding/Wash Buffer.
9. Place the spin column onto a new 1.5ml microcentrifuge tube. Add 200μl of HisLink™ Elution Buffer. Cap the spin column and tap or flick it several times to resuspend the resin. Wait 3 minutes.
   Note: HQ-tagged proteins may elute with a lower concentration of imidazole (50–100mM) compared to polyhistidine-tagged proteins. Section 4.A has details on the use of imidazole for elution.
10. Centrifuge the spin column and microcentrifuge tube at 14,000rpm for 1 minute to collect the eluted protein.

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RNA Extraction with Pure Yield™RNA Midiprep System (Promega)

Sample Preparation(E.coli)

Materials to Be Supplied by the User

• TE buffer
• lysozyme(0.4 mg Lysozyme/ mL TE buffer)
• Lysis Solution(20 µL β-Mercaptoethanol/ mL RNA Lysis Buffer)
• RNA Wash Solution(206 mL RNA Wash Solution + 350 mL 95% ethanol)

1. Inoculate 10ml of growth medium with a single colony of your bacterial sample. Incubate overnight at the appropriate temperature with shaking. Do not use the overnight culture directly for RNA isolation; instead use it to inoculate a fresh culture as described in Step 2.
2. Dilute the overnight culture 1:50 and grow to an optical density (O.D.600) of 0.6–1.0 by spectrophotometry. This should only take a few hours. If growth is too slow, reduce the dilution factor by adding a larger volume of inoculum to fresh medium.
   Note: The doubling time of E. coli is approximately 20 minutes in rich medium. The growth rate depends on the growth medium, temperature, strain and degree of oxygenation. For most purposes, the culture should be harvested in mid-log phase. A stationary phase E. coli culture generally contains 1–2 × 109 cells/ml, depending on the culture medium and aeration.
3. Transfer a volume of culture containing a maximum of 1 × 1010 cells to a centrifugal tube. Centrifuge at 5,500 × g for 5 minutes at 4°C. Carefully remove the supernatant, leaving the cell pellet as dry as possible.
4. Resuspend the cell pellet in 1ml of freshly prepared TE buffer containing lysozyme. Do not exceed 1ml per prep. Tap gently to mix.
   Note: For Gram-positive bacteria use 3mg/ml lysozyme; for Gram-negative bacteria, use 0.4mg/ml lysozyme.
5. Incubate the resuspended cells at room temperature. For Gram-positive bacteria, incubate the cells for 5–10 minutes; for Gram-negative bacteria, incubate the cells for 3–5 minutes.
6. Add 1ml of Lysis Solution containing BME. Vortex vigorously until lysis is complete and no clumps are present. Incubate on ice for 10 minutes to complete the lysis.
   Optional: Homogenize the lysate at high speed, using a rotor-stator homogenizer (such as a BioSpec Tissue-Tearor™), until no visible clumps remain.
7. Proceed to Lysate Clearing.

Lysate Clearing

1. Transfer 2ml of lysate to a 15ml, capped, disposable centrifugal tube. Lysate volumes larger than 2ml may be processed with multiple columns or the remainder can be stored at –70°C for later use. If the volume of lysate is not a multiple of 2ml, add an appropriate volume of Lysis Solution to achieve 2ml per prep.
2. Add 4ml of RNA Dilution Buffer (blue) to 2ml of lysate. Immediately seal the tube, and mix thoroughly by inverting the tube 3–4 times and then vortexing. Process all samples before continuing.
3. Thoroughly mix the white Clearing Agent by shaking or vortexing the bottle until all of the material is in suspension. Mix until no material adheres to the bottom of the bottle when inverted. This may take several minutes.
   Note: The Clearing Agent settles over time and should be remixed between samples. When the Clearing Agent is mixed properly, it will not clog pipet tips.
4. Add 1ml of Clearing Agent to the diluted lysate. Immediately seal the tube, and mix by inverting 2–3 times and vortex until homogeneous. Process all samples before continuing.
   Notes:
   1. Combining the RNA Dilution Buffer and Clearing Agent prior to use is not recommended. The Clearing Agent settles more quickly when diluted, and the mixture thickens over time.
   2. The Clearing Agent may form a white powder on the threads of the bottle. You can collect the powder with a wet paper towel and discard it as nonhazardous waste.
5. Place the tubes in a heating block or water bath at 70°C. Make sure that the tubes are spaced such that they are fully in contact with the heating block or water and the entire sample is heated. Incubate at 70°C for 5 minutes to denature the samples. The heated mixtures may form clumps of precipitated debris.
   [!] Incomplete heating of samples may result in DNA contamination.
6. Remove the tubes and cool the heated samples at room temperature for at least 5 minutes.
7. Wearing clean gloves, place one blue PureYield™ Clearing Column for each sample in a 50ml collection tube. Save the collection tube caps. Label the Clearing Columns and collection tubes to maintain sample identity
   Note: If cross-contamination is a concern, flat-top tubes should be used, as they can be sealed during centrifugation.
8. Mix each sample by vortexing or vigorously shaking until homogeneous. Immediately pour the mixture into the assembled PureYield™ Clearing Column/collection tube. If you are using a flat-top tube, you can seal the assembled column with the cap if desired. Process each sample individually.
   [!]The flowthrough from the blue PureYield™ Clearing Column must be collected in a collection tube by centrifugation. Vacuum cannot be used.
9. Centrifuge the PureYield™ Clearing Column assembly in a swinging bucket rotor at 2,000 × g at 22–25°C for 10 minutes to clear the lysate.
   Note: The Clearing Agent and sample debris will form a solid cake on the surface of the Clearing Membrane. The color may vary, depending on the sample type. If liquid remains on the surface of the debris cake, centrifuge for an additional 5–10 minutes at a higher g force (e.g., 2,500–3,000 × g). A blue-green cleared lysate will collect in the 50ml collection tube.
   [!]Do not discard the cleared lysate in the collection tube. RNA is present in the blue-green cleared lysate.
10. Discard the blue Clearing Column, and save the cleared lysate in the collection tube. If a debris pellet is found in the bottom of the collection tube, transfer the cleared lysate to a fresh 50ml tube.
11. Proceed to RNA Purification by Centrifugation (Spin)

RNA Purification by Centrifugation (Spin)

1. Wearing clean gloves, place a clear PureYield™ Binding Column in a 50ml collection tube for each sample. Save the collection tube caps. Label the columns and tubes to maintain sample identity.
   Note: If cross-contamination is a concern, flat-top tubes should be used, as they can be sealed during centrifugation.
2. Add 4ml isopropanol to the cleared lysate and mix thoroughly by swirling or capping and shaking the tubes. Vortexing is not recommended. Immediately pour the mixture into the PureYield™ Binding Column/collection tube assembly (Step 1). Cap the tube if desired.
3. Centrifuge at 2,000 × g for 10 minutes in a swinging bucket rotor to capture the RNA.
4. Carefully remove the PureYield™ Binding Column from the collection tube and discard the flowthrough. Return the Binding Column to the tube.
5. Verify that ethanol has been added to the RNA Wash Solution. Add 20ml of RNA Wash Solution to the PureYield™ Binding Column. Cap the tube if desired. Centrifuge at 2,000 x g for 5 minutes.
6. Empty the Collection Tube as in Step 4. Repeat the wash in Step 5 with an additional 10ml of RNA Wash Solution, increasing the centrifugation time to 10 minutes to empty the column and dry the Binding Membrane.
   Optional: To further reduce ethanol carryover, empty the collection tube and centrifuge the PureYield™ Binding Column for an additional 5 minutes at 2,000 × g.
7. Carefully transfer the PureYield™ Binding Column to a fresh 50ml collection tube. Save the cap for the elution step. Use care to make sure that the flowthrough does not contact the column.
   Note: If the PureYield™ Binding Column is contacted by the flowthrough, empty the collection tube and centrifuge the assembly for 1 minute before transferring the Binding Column to a fresh tube (Step 7).
8. Add 1ml Nuclease-Free Water to the PureYield™ Binding Column, preferably using an ART® Aerosol Resistant Tip. Be careful to completely cover the surface of the membrane with the water. If desired, cap the tube with the clean cap. Incubate at room temperature for 2 minutes to release the RNA into solution. Centrifuge at 2,000 x g for 3 minutes to collect the RNA.
   Note that with some types of centrifuge braking and vacuum systems, some upward displacement of the membrane can occur during the elution step. This does not affect the yield or performance of the system.
9. Remove the PureYield™ Binding Columns and discard. Store the aliquots of the purified RNA at –70°C using sterile, low-binding (i.e., siliconized), RNase-free microcentrifuge tubes.
   Note: Ethanol contamination may occur with the centrifugation (spin) format. If the RNA sample is not heat denatured prior to gel loading, it may float out of the well. If this occurs, leave a portion of the sample open to the air for 10 minutes to allow the ethanol to evaporate. The RNA sample may also be incubated at 37°C to increase the evaporation rate.

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Western blotting

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