Taq polymerase PCR

1. Thaw the PCR Master Mix
   Vortex the Master Mix and then spin it briefly in a microcentrifuge to collect the material in the bottom of the tube.
2. Prepare the following reagent mixes.

For a 20 μl reagent volume:

Component	Volume (μl)
GoTaq® Green Master Mix	10
10 µM Primer (F)	0.5
10 µM Primer (R)	0.5
100 ng/µl DNA template	0.5
ddH2O	8.5
Total	20

3. Denaturation
   Generally, a 5-minute initial denaturation step at 95℃ is sufficient. Subsequent denaturation steps will be between 30 seconds and 1 minute.
4. Annealing
   Optimize the annealing conditions by performing the reaction starting approximately 5℃ below the calculated melting temperature of the primers and increasing the temperature in increments of 1℃ to the annealing temperature.The annealing step is typically 30 seconds to 1 minute.
5. Elongation
   The extension reaction is typically performed at the optimal temperature for Taq DNA polymerase, which is 72–74℃.
   Allow approximately 1 minute for every 1kb of DNA to be amplified.
   A final extension of 5 minutes at 72–74℃ is recommended.
6. Refrigeration
   If the thermal cycler has a refrigeration or ”soak” cycle, the cycling reaction can be programmed to end by holding the tubes at 4℃ for several hours.
   This cycle can minimize any polymerase activity that might occur at higher temperatures, although this is not usually a problem.
7. Cycle Number
   Generally, 25-30 cycles result in optimal amplification of desired products. Occasionally, up to 40 cycles may be performed, especially for detection of low-copy targets.

Denaturation	95℃	3 min	40 X cycle
	95℃	30 s
Annealing	58℃	30 s
Elongation	72℃	1 min per kb
	72℃	7 min
Refrigeration	4℃	∞

Pfu polymerase PCR

1. Gently mix the reagents and collect the liquid at the bottom of the tube with a quick spin.
2. Transfer reagent quickly to a preheated thermocycler (98℃).

For a 25 μl reagent volume:

Component	Volume (μl)
5X Phusion HF or GC buffer	5
10 mM dNTPs	0.5
10 µM Forward Primer	1.25
10 µM Reverse Primer	1.25
Template DNA	variable
Phusion DNA Polymerase	0.25
DMSO (optional)	(0.75)
Nuclease-Free Water	to 25
Total	25

Intial Denaturation	98℃	30 s
25-35 cycles	98℃	5-10 s
	45-72℃	10-30 s
	72℃	15-30 s/kb
Final Extension	72℃	5-10 min
Hold	4℃	∞

PCR cleanup

1. Sample Preparation
   • Transfer up to 100 μl of reagent product to a 1.5 ml microcentrifuge tube.
   • Add 5 volumes of Gel/PCR Buffer to 1 volume of the sample and mix by vortex.
2. DNA Binding
   • Place a DFH Column in a 2 ml Collection Tube.
   • Transfer the sample mixture to the DFH Column. Centrifuge at 14-16,000 xg for 30 seconds. Discard the flow-through and place the DFH Column back in the 2 ml Collection Tube.
3. Wash
   • Add 600 μl of Wash Buffer (make sure ethanol was added) into the center of the DFH Column. Let stand for 1 minute at room temperature. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through and place the DFH Column back in the 2 ml Collection Tube.
   • Centrifuge at 14-16,000 x g for 3 minutes to dry the column.
4. DNA Elution
   • Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube.
   • Add 20 μl of (60-70℃) pre-heated ddH2O into the center of the column matrix. Let stand for at least 2 minutes.
   • Centrifuge for 2 minutes at 14-16,000 x g to elute the purified DNA.

TA-ligation

1. Prepare the following components.

Component	Volume (μl)
insert : vector (volume ratio) = 7 : 1 (or 4 : 4)	8
T4 Ligase (NEB)	1
10X Ligase buffer (NEB)	1
Total	10

2. Set at room-temperature for 3 hrs or 16℃ overnight for ligation

Target vector ligation

1. Prepare the following components.

Component	Volume (μl)
insert : vector (mole ratio) = 3 : 1 (or 1 : 1)	8
T4 Ligase (NEB)	1
10X Ligase buffer (NEB)	1
Total	10

2. Set at room-temperature for 3 hrs or 16℃ overnight for ligation.

Transformation

1. Thaw competent cells with room temperature or water bath for 10~20 seconds until ⅓ thawed or thaw competent cells with the ice bucket.
2. Mix 10 μl ligation product with 30 μl competent cells by vortexing 1 second.
3. Incubate on ice for 5 minutes.
4. Transfer to LB plate with desired antibiotics at 37℃, and spread transformed cells by rolling glass beads.
5. Incubate the plate at 37℃ for 16~18 hours.

Colony PCR

1. Preparation
   • Prepare 200 μl tubes and 1.5 ml tubes.
   • Add 5 μl ddH2O into each 200 μl tube.
   • Add 50 μl TB + antibiotic into each 1.5 ml tube.
2. Pick colony
   • Pick a single colony by toothpick into each tube, stir with 5 μl ddH2O, and then stir with 50 μl TB + antibiotic.
3. Prepare PCR mix
   • The PCR mix needs to prepare (n+1) tubes every 15 tubes. (15+1, 30+2, 45+3, ...)

Component	Volume (μl)
GoTaq® Green Master Mix	10
10 µM Primer (F)	0.5
10 µM Primer (R)	0.5
ddH2O	4
Total	15

4. Make mixture
   • Add 15 μl PCR mix to the 200 μl tube. ( total 20 μl )
5. Run PCR

Denaturation	95℃	3 min
	95℃	30 s	40 X cycle
Annealing	58℃	30 s
Elongation	72℃	1 min per kb
	72℃	7 min
Refrigeration	4℃	∞

Plasmid extraction

1. Harvesting
   • Transfer 1.5 ml of cultured bacterial cells (1-2 x 10⁹ E. coli grown in TB medium) to a 1.5 ml microcentrifuge tube.
   • Centrifuge at 5000 x g for 3 minute at room temperature to form a cell pellet then discard the supernatant completely.
   • Repeat the harvesting step as required for samples between 1.5-6.0 ml using the same 1.5 ml microcentrifuge tube.
2. Resuspension
   • Add 200 μl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge tube containing the cell pellet.
   • Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved.
3. Cell Lysis
   • Add 200 μl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times.
   • Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.
4. Neutralization
   • Add 300 μl of PD3 Buffer then mix immediately by inverting the tube 10 times.
   • Do not vortex to avoid shearing the genomic DNA.
   • Centrifuge at 14-16,000 x g for 3 minutes at room temperature.
   • If using >5 ml of bacterial cells,centrifuge at 16-20,000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection Tube.
5. DNA Binding
   • Transfer all of supernatants to the PDH Column. Use a narrow pipette tip to ensure the supernatant is completely transferred without disrupting the white precipitate.
   • Centrifuge at 14-16,000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
6. Wash
   For Improved Downstream Sequencing Reactions
   • Add 400 μl of W1 Buffer into the PDH Column.
   • Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube. Proceed with Wash Buffer addition.
   For Standard Plasmid DNA Purification
   • Add 600 μl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column.
   • Centrifuge at 14-16,000 x g for 30 seconds at room temperature. Discard the flow through then place the PDH Column back in the 2 ml Collection Tube.
   • Centrifuge at 14-16,000 x g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PDH Column to a new microcentrifuge tube.
7. Elution
   • Add 50 μl elution buffer and centrifuge at 14-16,000 x g for 2 min.
   • Save the elute.

Digestion

1. digest insert

Component	Volume (μl)
insert	43
10X buffer	5
restriction enzymes	1 + 1
Total	50

2. digest target vector

Component	Volume (μl)
vector	3 μg
10X buffer	3
restriction enzymes	0.3 + 0.3
ddH2O	to 30
Total	30

Gel extraction

1. Gel Dissociation
   • Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice.
   • Transfer up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube.
   • Add 500 μl of Gel/PCR Buffer and mix by vortex. Incubate at 55-60℃ for 10-15 minutes to ensure the gel slice has been completely dissolved.
   • During incubation, invert the tube every 2-3 minutes. If the color of the mixture has turned to purple, add 10 μl of 3M Sodium Acetate (pH5.0) and mix thoroughly.
   • Cool the dissolved sample to room temperature.
2. DNA Binding
   • Place a DFH Column in a 2 ml Collection Tube.
   • Transfer 800 μl of the sample mixture to the DFH Column.Centrifuge at 14-16,000 xg for 30 seconds. Discard the flow-through and place the DFH Column back in the 2 ml Collection Tube.
   • If the sample mixture is more than 800 μl, repeat the DNA Binding step.
3. Wash
   • Add 400 μl of W1 Buffer into the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube.
   • Add 600 μl of Wash Buffer (make sure ethanol was added) into the DFH Column. Let stand for 1 minute at room temperature. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube.
   • Centrifuge at 14-16,000 x g for 3 minutes to dry the column matrix.
4. DNA Elution
   • Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube.
   • Add 20 μl of (60-70℃) pre-heated Elution Buffer or TE into the center of the column matrix. Let stand for at least 2 minutes to ensure the Elution Buffer is completely absorbed.
   • Centrifuge for 2 minutes at 14-16,000 x g to elute the purified DNA.

Protein induction

1. Preparation
   • Cultured 3 ml bacteria in TB-antibiotics at 37℃ overnight.
2. Refresh
   • Quantify and dilute the grown bacteria to OD600 value around 0.02.
   • Refresh the diluted bacteria at 37℃ for 3 hrs.
   • Quantify the OD600 value of refreshed bacteria. If OD600 value reaches 0.2~0.4, store 1 ml of refreshed bacteria as the non-induction sample.
3. Induction
   • Induced the protein expression in the rest of refreshed bacteria by 1mM IPTG at 37℃ for 4 hrs.
   • Quantify the OD600 value of induced bacteria. If OD600 reaches 0.2~0.4, save all bacteria as the induction sample.
4. Storage
   • Non-induction and induction samples are centrifuged at 5000 rpm for 3 mins.
   • Remove the supernatant.
   • Directly store the pellet in -20℃, or lyse the pellet with 100μl 1x sample buffer and store at -20℃.

Protein extraction

1. Pellet bacterial cells by centrifugation at 5000 x g for 10 minutes. Weigh biomass and proceed to Step 2 or freeze biomass at -20℃ to -80℃.
2. Warm required amount of B-PER Complete Reagent (5ml reagent/g of biomass) to room temperature.

3. Add 5ml of B-PER Complete Reagent per gram of cell pellet. Pipette the suspension up and down until it is homogeneous.
4. Incubate 15 minutes at room temperature with gentle rocking.
5. Centrifuge lysate at 16,000 x g for 20 minutes to separate soluble proteins from the insoluble proteins.

Protein quantification

1. Prepare dye reagent by diluting 1 part Dye Reagent Concentrate with 4 parts DDI water. Filter through a Whatman #1 filter (or equivalent) to remove particulates. This diluted reagent may be used for about 2 weeks when kept at room temperature.
2. Prepare three to five dilutions of a protein standard, which is representative of the protein solution to be tested. The linear range of this microtiter plate assay is 0.05 mg/ml to approximately 0.5 mg/ml. Protein solutions are normally assayed in duplicate or triplicate.
3. Pipet 10 μl of each standard and sample solution into separate microtiter plate wells.
4. Add 200 μl of diluted dye reagent to each well. Mix the sample and reagent thoroughly using a microplate mixer. Alternatively, use a multi-channel pipet to dispense the reagent. Depress the plunger repeatedly to mix the sample and reagent in the well. Replace with clean tips and add reagent to the next set of wells.
5. Incubate at room temperature for at least 5 minutes. Absorbance will increase over time; samples should incubate at room temperature for no more than 1 hour.
6. Measure absorbance at 595 nm.

SDS-PAGE

Fixing solution
Ethanol	150 ml
Glacial acetic acid	50 ml
ddH2O	300 ml
Total	500 ml

Staining solution
Methanol	150 ml
Glacial acetic acid	50 ml
Coomassie brilliant blue	1 g
ddH2O	300 ml
Total	500 ml

Distaining solution
Methanol	150 ml
Glacial acetic acid	50 ml
ddH2O	300 ml
Total	500 ml

Procedure

1. Gel preparation
   1. Wipe glass plates and spacers and assemble in gel casting apparatus.
   2. Mix components for the resolving gel and pour into the gel plate.
   3. Add 200 μl ddH2O on the top of the resolving gel to flat resolving gel.
   4. Until resolving gel solidified, clean up the ddH2O on the resolving gel.
   5. Mix components for the stacking gel, and pour into the gel plate.
   6. Insert comb into the top of the spacers, and then wait for the gel to solidify.
2. Gel electrophoresis
   1. Add 1x running buffer to the running tank and put the cast gel into it.
   2. Load 10 μl marker into the first well, and load 20 μl protein samples per well.
   3. The gel will run at 80 V until the dye front is migrated into the running gel (about 20 minutes), and increase to 100 V until the dye front reaches the bottom of the gel (about 90 minutes).
3. Gel fixing, staining and distaining
   1. Put gel into gel-fixing solution for 1 hour.
   2. Stain the gel with staining solution for 20 minutes.
   3. Distain the gel for about 1.5~2 hours until the bands are clear enough.

Bacteria stock

1. Add 500 μl of the overnight culture to 500 μl of 50% glycerol in a 1.5 ml tube (1:1) .**Make the 50% glycerol solution by diluting 100% glycerol in ddH2O.
2. Freeze the glycerol stock tube at -80℃.

Recipe for LB

Component
ddH2O	1 L
Bacterial Agar	15 g
LB Broth	20 g
Ampicillin/Kanamycin/Chloramphenicol	500 μl

**Before adding antibiotics, sterilized the solution for an hour.

Recipe for TB

Component
ddH2O	1 L
Terrific Broth	47.6 g
100% glycerol	4 ml
Ampicillin/Kanamycin/Chloramphenicol	500 μl

**Before adding antibiotics, sterilized the solution for an hour.

Recipe for ampicillin

Component
Sterilized ddH2O	10 ml
Ampicillin powder	1 g

**Allocate 10 microcentrifuge tubes, using a syringe and filter.

Recipe for kanamycin

Component
Sterilized ddH2O	10 ml
Kanamycin powder	0.5 g

**Allocate 10 microcentrifuge tubes, using a syringe and filter.

Recipe for chloramphenicol

Component
Sterilized ddH2O	10 ml
Chloramphenicol powder	0.25 g

**Allocate 10 microcentrifuge tubes, using a syringe and filter.

Recipe for TAE buffer

Component	Volume
50x TAE	20 ml
ddH2O	980 ml
Total	1 L

Recipe for TBE buffer

Component	Volume
10x TAE	100 ml
ddH2O	900 ml
Total	1 L

Recipe for 0.8% gel preparation

1. Mix 1.6 g agarose with 200 ml 1x TAE, and boil in a microwavable flask by microwave for 1-3 min, until the agarose is completely dissolved.
2. Cool down the agarose solution for a while.
3. Add ethidium bromide (EtBr) to the agarose solution.
4. Pour the agarose into a gel tray with the well comb in place.
5. Place at room temperature for 20-30 mins, until it has completely solidified.

Recipe for 2% gel preparation

6. Mix 4 g agarose with 200 ml 1x TAE, and boil in a microwavable flask by microwave for 1-3 min, until the agarose is completely dissolved.
7. Cool down the agarose solution for a while.
8. Add ethidium bromide (EtBr) to the agarose solution.
9. Pour the agarose into a gel tray with the well comb in place.
10. Place at room temperature for 20-30 mins, until it has completely solidified.