T4 phage Multiplication with E. coli

Materials

• E. coli (K-12 W3110)
• LB medium (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L NaCl)
• Agarose
• Enterobacteria T4 phage purchased from NBRC (Search number:20004)
• Rehydration fluid
• Dimethyl sulfoxide (DMSO) or glycerol

The day before

A. Making culture solution

1. Thaw E. coli (K-12 W3110) stored at -80 °C.
2. Scrape the germ with platinum loop in a clean bench and then 5 mL of autoclaved LB medium in a test tube.
3. Do shaking culture overnight.

B. Making medium

1. Make 120 mL of LB medium and then pour 4 mL of LB medium each into 4 test tubes.
2. Add 0.028 g (concentration 0.7 %) of agarose each to 4 test tubes (step 1). [for top agar]
3. Add 1.5 g (concentration 1.5 %) of agarose to about 100 mL of LB medium (step 1). [for bottom plate]
4. Autoclave the media.

The day

Note: Keep on a germicidal lamp in a clean bench for 5 minutes.

1. Add 0.3 mL of the culture solution (Section 1.A) to 3 mL of LB medium and then shake it for 3-4 hours.
   Note: The shaking time depends on a growth rate of the host.
2. Melt slowly the medium for bottom plate (Section 1.B step 3) with a microwave oven. Dispense 15 mL of it each to 5-6 plates and then incubate them at 45 °C.
3. Melt slowly the top agars (Section 1.B step 2) with a microwave oven and then incubate them at 45 °C.
4. Add 0.2 mL of the E. coli culture solution (Section2.B step 1) each into the top agars and then mix them lightly. Pile up the top agar on the bottom plate. Stand them for them to go solid (for about 20 minutes) [ phage culture plate ]
5. Add 200 µL of the special rehydration fluid to T4 phage in an ampule in a clean bench and then stand for a few minutes.
6. Mix mildly with a micropipette and then transfer it to a sterilized tube. Add 0.8 mL of the rehydration fluid to it. [ phage solution ]
7. Apply 250 µL of the phage solution each on 4 phage culture plates. Expand the solution on a surface of the plate and then culture it.
   Note: Let a part of the surface of a phage culture plate alone.
   Reason: Discriminate phage proliferation by a difference of E. coli growth rates between the area that T4 phage was swabbed on and the area that T4 phage was not swabbed on, for the phage kills E. coli.
8. Culture the phage at 37 °C overnight. [phage plate]
   Note: Store the surplus bottom plate with wrapped in aluminum foil.

The next day

Note: Keep on a germicidal lamp in a clean bench for 5 minutes.

1. Add 1 mL of liquid culture medium to the phage plate. Collect the top agar and then transfer it to 15 mL precipitation tube for centrifugal separation. Add 1 mL of liquid culture medium into the tube and then wash and collect the surplus top agar.
   Note: If there are plural plates, transfer the top agar of 2 plates to 1 tube.
2. Vortex it lightly and then transfer 800 µL of the solution and agar each to a 1.5 mL tube.
3. Centrifuge (10,000 rpm 15 min 4 °C) the tubes.
4. Collect the supernatant and then transfer it to a syringe.
5. 5. Attach a filter (the pore size is 0.2 µm). Transfer 1 mL of the supernatant each to a new 1.5 mL tube. Filtrate contains the phage (size: 25~200 nm).
6. Melt the culture for bottom plate with a microwave oven and then incubate it at 45 °C
7. Add 0.2 mL of culture solution of E. coli to the culture (step 6) and then pour it into a plate.
8. Drop a small amount of filtrate (step 5) on a plating medium of E. coli.
9. Culture it overnight. If the proliferation of E. coli is not seen on the spot (step 8), determine that the sterilization treatment is positive.

DNA Extraction from T4 phage

Materials

Filtrate of T4 phage	800 µL
13% PEG solution (with 0.8M NaCl)	400 µL
20 mM Tris-HCl-buffer	100 µL
sterile water
phenol chloroform mixture	200 µL
1% SDS	20 µL
500 mM EDTA solution	4 µL
Isopropanol	250 µL
70% ethanol	300 µL
TE buffer	50 µL

Method

Note: Keep on a germicidal lamp in a clean bench for 5 minutes.

1. Transfer 800 µL of the filtrate of phage and 400 µL of 13% PEG solution to a 1.5 mL tube and then leave it to stand on the ice for 30 minutes.
2. Centrifuge (14,000 rpm, 4 °C, 10 minutes) it and then discard supernatant (autoclave).
3. Add 100 µL of 20mM Tris-HCl-buffer to the precipitation and then mix it.
4. Add 100 µL of phenol chloroform mixture to the solution (step 3) and then mix with inversion it 10 times.
5. Centrifuge (14,000 rpm, room temperature, 5 minutes) it and then transfer the supernatant to another 1.5 mL tube.
   Note: Don’t suck white middle layer.
6. Add 20 µL of 1% SDS to the solution. Add 4 µL of 500 mM EDTA and then mix it sufficiently. Stand it at room temperature for 20 minutes.
7. Add 100 µL of phenol chloroform mixture to the solution (step 6) and then mix with inversion it 10 times.
8. Centrifuge (14,000 rpm, room temperature, 5 minutes) it and then transfer the supernatant to another 1.5 mL tube.
9. Add 250 µL of isopropanol and then stir it mildly.
10. Centrifuge (14,000 rpm, 4°C, 5 minutes) it and then discard the super natant (autoclave).
11. Add 300 µL of 70% ethanol and then mix mildly and wash the precipitation.
12. Centrifuge (14,000 rpm, room temper, 5 minutes) it and then discard the super natant.
13. Remove ethanol completely at 47 °C. Add 50 µL of TE buffer and then store at 4 °C.

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.

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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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.

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.

Circular RNA Detection

1. RNase processing: to find the circular mRNA
2. RT-PCR: to synthesize cDNA and to detect the cDNA synthesized from circular mRNA or endogenous RNA
3. Electrophoresis: to detect the DNA synthesized from the cDNA

1. RNase processing

Mix the following reagents.

	Group1(RNaseA)	Group2(RNaseR)
RNaseA	1 µL
RNaseR		1 µL
buffer		1 µL
RNA solution	9 µL	8 µL
total	10 µL	10 µL

Incubate them at 37 °C for 20 minutes.

2.RT-PCR

Mix the following reagents.

	Group1(RNaseA)	Group2(RNaseR)	Group3(non-treated)
RNA solution(after RNase processing)	8 µL	8 µL
RNA solution			6 µL
pure water			2 µL
Oligo(dT)15primer	1 µL	1 µL	1 µL
Random primer	1 µL	1 µL	1 µL
total	10 µL	10 µL	10 µL

Incubate them at 70 °C for 5 minutes.
Incubate them at 4 °C for 5 minutes.
Incubate them on ice.
Mix the following reagents.

Nuclease Free Water	4.58 µL
GoScript™ 5×reaction buffer	12.2 µL
25mM MgCl2	6.1 µL
10mM PCR Nucleotide Mix	3.05 µL
Recombinant RNasin Ribo nuclease Inhibitor	1.52 µL
GoScript™ Reverse	3.05 µL

Add 10 µL of it each to group1,2,3.
Synthesize cDNA.

Annealing	At 25 °C for 5 minutes
Elongation	At 42 °C for 60 minutes
Inactivation	At 70 °C for 15 minutes
	At 4 °C for ∞

Mix the following reagents.

• Group1:RNaseA processing
• Group2:RNaseR processing
• Group3:without RNase processing
• Group4:total RNA without RNase processing and reverse transcription

	Group1		Group2		Group3		Group4
	1	2	3	4	5	6	7	8
Nuclease Free Water	10 µL	10 µL	10 µL	10 µL	10 µL	10 µL	10 µL	10 µL
GoTaq 2×mix	25 µL	25 µL	25 µL	25 µL	25 µL	25 µL	25 µL	25 µL
primer Fw (to detect circular mRNA)		2.5 µL		2.5 µL		2.5 µL		2.5 µL
primer Rv (to detect circular mRNA)		2.5 µL		2.5 µL		2.5 µL		2.5 µL
primer Fw (to detect linear(endogenous) mRNA)	2.5 µL		2.5 µL		2.5 µL		2.5 µL
primer Rv (to detect linear(endogenous) mRNA)	2.5 µL		2.5 µL		2.5 µL		2.5 µL
cDNA solution (Group1)	10 µL	10 µL
cDNA solution (Group2)			10 µL	10 µL
cDNA solution (Group3)					10 µL	10 µL
RNA solution (Group4)							10 µL	10 µL

Do the PCR assay.

At 94°C for 3 minutes
At 94°C for 20 seconds	35 cycles
At 58°C for 20 seconds
At 72°C for 50 seconds
At 72°C for 70 seconds
At 4°C for ∞

3.Electrophoresis

Western blotting

Activation of PVDF membrane

1. Cut out PVDF membrane (6 cm × 9 cm per a gel).
2. Immerse the PVDF membrane in 100%EtOH for 5 minutes.
3. Cut out filter paper (6 cm × 9 cm × 6 sheets per a gel).
4. Superpose in order of three pieces of filter paper, a PVDF membrane and three pieces of filter paper.
5. Immerse it in transfer buffer.