Team:IISER-Pune-India/Experiments

Gibson
RF Cloning
Expression
6xHis-tag
Strep-tag
SDS-PAGE
Ni-NTA
IC50
RBC Invasion Assay
PCRs
Site Directed Mutagenesis

Gibson's Assembly Protocols

Supplies

  • 15 μL aliquots or a 2X stock of Gibson master mix
  • Tubes in both a 96 well holder
  • 50 mL tube
  • Sterile, nuclease-free water
  • Positive control: Positive Control DNA Mix (see below)
  • Primer Design using Gibson

Protocol

  1. Use PCR to produce the DNA segments needed for assembling the new construct. Generally, it is best to use a high fidelity polymerase, such as Phusion, to amplify the Gibson fragments.
  2. Confirm the success of each PCR by running 5 μL of the reaction on an agarose gel.
  3. Mix 10 ng-100 ng of each of the DNA fragments together (such that their ratios are equimolar) into a total volume of 5 μL.
  4. If using the 2X Gibson Master Mix from NEB, add 10 μL of total DNA (containing all the fragments) to 10 μL of the mix. If using the homemade Gibson mix (refer to the recipe at the bottom of this page), add 5 μL of DNA to 15 μL of mix. Be careful with pipetting small volumes.
  5. Mix well by pipetting.
  6. Incubate the reaction at 50°C for 1 hour. For each Gibson reaction, perform two transformations.
  7. Perform a 1:4 dilution of the Gibson reaction in nuclease-free water, add 4 μL to 50 μL of chemically competent cells.
  8. Add 4 μL of the Gibson reaction to 50 μL competent cells without dilution.
    (Note: If electroporating, dilute the reaction 1:5 in water or PCR purify the reaction prior to transformation.)
  9. Follow transformation protocol for chemically competent cells.
  10. Run an aliquot of the final reaction on a gel to verify the presence of your construct. It may be useful to make a 'no incubation' control to determine if the chemistry of the reaction happened.

Expected Results

The positive control should yield ampicillin resistant colonies when transformed into E. Coli. Depending on the number of fragments in the reaction, different efficiencies will be observed. Gibson reactions with a higher number of fragments tend to be less efficient, therefore there will likely be few colonies on the plates.

Positive Control

Competency of cell was tested by two fragment assembly of pUC 19 plasmid. Positive control mix is obtained by PCR amplification of Fragment 1 and Fragment 2.

Use the following reaction mixture

32.7 μL dH2O
10 μL 5X HF Buffer
1 μL 10uM dNTP
2.5 μL 10uM FW primer
2.5 μL 10uM RV primer
1 μL ~3ng/μL pUC19 template
0.3 μL Phusion Polymerase

Use the following PCR program for both F1 and F2 PCRs

98°C 0:30
58°C 0:30
72°C 0:45
Go to step 2, 25X
72°C 10:00
  1. After the reaction is completed, add 1 μL of Dpn1 (20,000 U/mL) to each reaction, briefly vortex and spin down the reactions, and then incubate in the PCR machine at 37°C for 30 minutes.
  2. To test the effectiveness of Dpn1, construct the above PCR reaction without Phusion polymerase, digest and purify it in parallel with the F1 and F2 PCRs, and transform it alongside the other reactions.
  3. Any ampicillin-resistant transformants produced from transforming this negative reaction indicate that the Dpn1 digest was ineffective at digesting the pUC19 template. The solution to this is to use fresh Dpn1 in the digest, or to gel extract F1 and F2 instead of using a Dpn1 digest and column purification to remove the pUC19 template.
  4. Test the success of the PCR by gel electrophoresis and purify the reaction using a PCR cleanup kit, and elute with buffer or water without EDTA.
  5. Quantify the concentration of F1 and F2 purifications.
  6. Combine F1 and F2 PCR products to a final concentration of 2.8ng/μL of each fragment.
  7. Dilute with the elution buffer used in the PCR purification if needed. This final mixture is the Positive Control DNA Mix.
  8. Using 5 μL of this reaction provides approximately 1ng/100bp of each fragment in the Gibson Assembly reaction.

References

1: Barrick Lab Protocol Manual

Outline

  • Design complementary hybrid primers.
    These primers should be complementary to both the desired insert and the destination vector.
  • ↓
  • Perform the first round of PCR.
    This creates a ‘mega-primer’ which comprises the insert sequence flanked by sequences complementary to the destination vector.
  • ↓
  • Perform the second round of PCR.
    In this the mega-primer initiates replication of the destination plasmid.
    [The new product obtained is an nicked circular plasmid.]
  • ↓
  • Selectively degrade parental vectors using DpnI.
  • ↓
  • Transform the product plasmid into competent cells.

Procedure

Buffer Compositions

  • Cold lysis buffer: 50 mM Tris,300 mM NaCl, 20 mM imidazole, pH 8.0
  • Elution buffer: 50 mM Tris, 600 mM imidazole, pH 7.0
  1. PCR
    1. PCR 1 (Mega Primer Formation)
      1. Run for 5 min at 95°C
      2. Repeat 35x:
        1. 30s at 95°C
        2. 30s at annealing temperature (as per gradient PCR)
        3. 1 min/1 kb template at 68°C
      3. Run for 10 min at 72°C
      4. Run PCR products on an agarose gel and purify it using a PCR purification kit.
      5. Dilute the final concentration to 100 ng/μl.
    2. PCR 2 (RF Cloning PCR)
      1. Run for 5 min at 95°C
      2. Repeat 35x:
        1. 30s at 95°C
        2. 30s at the temperature as per the Tm of the primers
        3. 1 min + 1 min/1 kb template at 68°C
      3. Run again for 10 min at 72°C.
      4. Run PCR products on an agarose gel.
      5. Digest the PCR products using REase DPN1 at 37℃ for 2 hrs.
      6. Transform the product into ultracompetent E.coli cells.
  2. Protein Expression
    1. Perform test expressions using BL21(DE3) cells induced with 1 mM IPTG in early log phase.
    2. Harvest 10 mL of cells, after 4h, at 37°C and lyse in a 600 μL cold lysis buffer, in the presence of lysozyme and DNaseI.
    3. Lyse cells by sonication and pellet cell debris for 7 min at 14 krpm, at 4°C.
    4. Wash supernatant bound to Ni2+-NTA spin column, 3 times with 600 μL cold lysis buffer and elute it in 200 μL elution buffer.
    5. To discriminate between protein insolubility and absence of expression, use a Ni2+-NTA spin column with the same buffers supplemented with 8 M urea.

References

1: Bond SR, Naus CC. RF-Cloning.org: an online tool for the design of restriction-free cloning projects. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W209-13. DOI: 10.1093/nar/gks396. Epub 2012 May 8. PMID: 22570410; PMCID: PMC3394257.

2: van den Ent F, Löwe J. RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods. 2006 Apr 30;67(1):67-74. DOI: 10.1016/j.jbbm.2005.12.008. Epub 2006 Feb 3. PMID: 16480772.

Expression

Reagents

Recommended Buffers/Solutions Concentration of Ingredients Notes
Kanamycin Stock solution: 50 µg/mL in H2O, sterile filtered Store in aliquots at -20°C
Buffer W
  • 100 mM Tris/HCl
  • 150 mM NaCl
  • 1 mM EDTA
  • Adjust pH to 8.0
LB Medium
  • 10 g/l trypton
  • 5 g/l yeast extract
  • 5 g/l NaCl
Glucose 20%, sterile filtered
IPTG Stock solution (1M): 238 mg/mL in H2O, sterile filtered Store in aliquots at -20°C
5x SDS-PAGE sample buffer
  • 0.250 M Tris/HCl
  • 25% glycerol
  • 7.5% SDS
  • 0.25 mg/mL bromophenolblue
  • 12.5 % v/v mercaptoethanol
  • Adjust pH 8.0

Equipment

  • Culture Plates
  • Pipettes
  • Glassware
  • Microfuge
  • Centrifuge

Protocol

  1. Preculture: Inoculate 2 mL of LB medium containing 50 μg/mL Kanamycin with a fresh colony harbouring the pET28a expression plasmid and shake overnight (200 rpm) at 37°C.
  2. Culture for expression: Inoculate 100 mL of LB medium containing 50 μg/mL Kanamycin with the preculture and shake at 37°C.
  3. Monitor the optical density at 550 nm (OD550). Cell suspension with an OD550 over 0.3 should be diluted with LB medium before measuring.
  4. Take a 1 mL sample immediately before induction. This sample is the non-induced control; pellet cells (microfuge, 30 seconds) and resuspend them in 80 μL Buffer W. Add 20 μL 5x SDS-PAGE sample buffer. Store at -20°C until SDS-PAGE analysis. The whole sample must be incubated in an ultrasonic bath for 15 minutes to shear the chromosomal DNA into small pieces and should be heated to 70°C for 10 minutes prior to SDS-PAGE.
  5. When OD550 equals 0.5-0.6, add 50 μL of IPTG stock solution (0.5 mM end concentration).
  6. Shake for 3 hours at 200 rpm.
  7. Harvest the cells by centrifugation at 4500 x g for 12 minutes (4°C). Add 5 μL 5x SDS-PAGE sample buffer to 20 μL supernatant and store at -20°C for SDS-PAGE analysis.

Troubleshooting

Problem Comments and Suggestions
No or low expression
  • Check the culture condition (e.g., IPTG, anhydrotetracycline, antibiotics).
  • Check vector (sequence, frame).
  • Check whether the protein is found in the insoluble fraction. Reduction of temperature during cultivation may solve this problem. (e.g., 16°C, 22°C, 26°C, 30°C)
  • Use another expression system. Use eukaryotic cells for expression (yeast, insect or mammalian cells).
Protein is degraded
  • Use protease deficient E. coli strains.
  • If degradation occurs during cell lysis, add protease inhibitor.
  • If the protein is small (<10 kDa), consider adding a terminal carrier protein.
  • Lowering the temperature during induction can reduce the problem.
  • Secretion of the recombinant protein to the periplasmic space can reduce the problem.
Protein is secreted Remove all signal sequences from the coding region.
Inclusion bodies are formed: protein is insoluble
  • Reduce expression level by modifying growth and induction conditions, e.g., lower culturing temperature (16°C, 22°C, 26°C, 30°C).
  • Use another expression system (e.g., Tet promoter instead of T7 promoter).

References

1. www.rsc.org/suppdata/cc/c0/c0cc01704c/c0cc01704c.pdf

Purification of the Proteins using 6xHistidine-tag

Reagents

Buffers/Solution Concentration of Ingredients
Ni-NTA lysis buffer
  • 20 mM HEPES (pH 7.4)
  • 150 mM NaCl
  • 1 mM PMSF
Lysozyme
5x SDS-PAGE sample buffer
  • 0.25 M Tris/HCl
  • 7.5% SDS
  • 25% glycerol
  • 0.25 mg/mL bromophenol blue
  • 12.5% v/v mercaptoethanol
  • Adjust pH 8.0
Ni-NTA Lysis Buffer
  • 20 mM HEPES (pH 7.4)
  • 150 mM NaCl
  • 1mM PMSF
Ni-NTA Wash Buffer
  • 20 mM HEPES (pH 7.4)
  • 150 mM NaCl
  • 20 mM imidazole
Ni-NTA Elution Buffer
  • 20 mM HEPES (pH 7.4)
  • 150 mM NaCl
  • 250 mM imidazole

Glasswares and Plastic wares

  • Pipettes and tips (including large orifice tubes)
  • Vortex mixer.
  • Distilled Water
  • Bench top shaker
  • Pipettes and tips, including large orifice tips for pipetting Antibody Bead.
  • 100 mL graduated cylinder and container.

Protocol

  1. Thaw the cell pellet for 15 minutes on ice and resuspend the cells in Ni-NTA Lysis Buffer at 2-5 mL per gram wet weight (typically, 30 mL is needed for a cell pellet from a litre of culture).
  2. Sonicate on ice-water mixture (1 on, 3 off, 60% amplitude for 10 mins).
  3. Centrifuge lysate at 30,000 x g for 30 minutes at 4°C to pellet the cellular debris.
  4. Add 5 μL 5x SDS-PAGE sample buffer to 20 μL supernatant and store at -20°C for SDS-PAGE analysis.
  5. Add 1 mL of the 50% Ni-NTA slurry to ~30 mL of cleared lysate (Ni-NTA beads washed and pre-equilibrated in Lysis Buffer) and mix gently by shaking (200 rpm on a rotary shaker) at 4°C for 60 min.
  6. Load the lysate/Ni-NTA mixture onto a PD10 column with capped bottom outlet.
  7. Remove bottom cap and collect the column flow-through.
  8. Wash extensively (~100 mL) with Ni-NTA Wash Buffer; collect wash fractions for SDS-PAGE analysis.
  9. Elute the protein 4 times with 2 mL each time with Ni-NTA Elution Buffer.
  10. Collect the eluate in four tubes and analyze by SDS-PAGE.

Troubleshooting

If the proteins don't bind to Ni-NTA

Problem Suggestions
His tag is not expressed properly Try moving His Tag to another terminal of protein. Try purification under denaturing conditions instead of native.
Degradation of His-tag after expression Avoid His-tag near the hotspot regions of inserted sequence (Part of protein under process).
Change in Binding conditions Composition and pH of all buffers should be checked prior to use. Dissociation of urea often causes a shift in pH. Proper concentration of imidazole. There should be no chelating agent in the buffer.

If the protein elutes in the Ni-NTA Wash Buffer

Problem Suggestions
Hidden His-tag Try purification under denaturing conditions. Lower washing stringency. Do not use imidazole in wash buffer.
Buffer Composition incorrect Check the pH and composition of the Ni-NTA buffer.
High washing stringency Increase the pH. Use low concentration of imidazole.

If the protein precipitates during purification

Problem Suggestions
Low temperature Purify proteins at room temperature
Aggregates of protein Try adding solubilization reagents such as 0.1% Triton X-100 or Tween-20, up to 20 mM β-mercaptoethanol, up to 2 M NaCl, or stabilizing cofactors such as Mg2+. These may be necessary in all buffers to maintain protein solubility.

If the protein does not elute

Problem Suggestions
Precipitation of proteins in column Elute under denaturing conditions. Perform binding and elution in Batch purification to maintain protein concentrations.
Mild elution conditions (Multimer of proteins) Elute with a pH of imidazole step-gradient to determine the optimal elution conditions.

If the proteins are contaminated or have low purity

Problem Suggestions
Column is large Use less Ni-NTA resin.
Less stringency in binding and washing conditions Increase the concentration of imidazole in the wash buffer to 20 mM. Use higher salt concentration (500 mM KCl) for wash.
Contaminants with tagged proteins Increase the detergent concentration to avoid nonspecific interactions. Add β-mercaptoethanol to reduce disulfide bonds. (<20mM)
Contaminants are truncated forms of the tagged protein Prevent protein degradation during purification by working at 4°C or by including protease inhibitors. Try purification with both His-tag and Strep-tag (2-step purification)

Discoloration of resin

Problem Suggestions
Less Ni ion concentration Ensure that there are no chelating compounds (resin color turns white) or reducing agents (resin color turns brown) present in all buffers.

References

1: www.iba-lifesciences.com/

2: Hochuli E, Döbeli H, Schacher A, 1987: J. Chromatogr. 411:177-184. New metal chelate adsorbent selective for proteins and peptide containing neighbouring histidine residues.

Purification of the Proteins using Strep-tag

Reagents

Buffers/Solution Concentration of Ingredients
Ni-NTA lysis buffer
  • 50 mM NaH2PO4
  • 300 mM NaCl
  • 10 mM imidazol
  • Adjust pH to 8.0
Buffer W
  • 100 mM Tris/HCl, pH 8
  • 150 mM NaCl
  • 1 mM EDTA
5x SDS-PAGE sample buffer
  • 0.25 M Tris/HCl
  • 7.5% SDS
  • 25% glycerol
  • 0.25 mg/mL bromophenolblue
  • 12.5% v/v mercaptoethanol
  • Adjust pH 8.0
Buffer E
Elution Buffer
  • 100 mM Tris/HCl, pH 8.0
  • 150 mM NaCl
  • 1 mM EDTA
  • 2.5 mM desthiobiotin

Glass wares and Plastic wares

  • Pipettes and tips (including large orifice tubes)
  • Vortex mixer
  • Distilled Water
  • Shaker
  • Pipettes and tips, including large orifice tips for pipetting Antibody Bead
  • 100 mL graduated cylinder and container

Protocol

  1. Chill Buffer W at 4°C.
  2. Take a 10 µl sample for analysis of the total protein content via SDS PAGE and/or Western blotting. 10 µl sample should be thoroughly mixed with 90 µl Buffer W and 25 µl of 5x SDS-PAGE sample buffer. Store it at -20 C.
  3. Sonicate the residual suspension under ice-cooling.
  4. If the lysate is very viscous, add RNase A (10 µg/mL) and DNase I (5 µg/mL) and incubate on ice for 10 – 15 min.
  5. Centrifuge the suspension at 13,000 rpm for 15 minutes at 4°C.
  6. Insoluble cell components are sedimented. If the recombinant protein forms inclusion bodies it will be present in the sediment.
  7. Carefully transfer the clear supernatant to a clean tube. For the analysis of insoluble part of the expressed proteins, dissolve the sediments with 1.25 mL 1x SDS-PAGE sample buffer with 1 mL Buffer W.
  8. Equilibrate the Strep-Tactin® column with 2 CVs (column bed volumes) Buffer W. Remove the first top cap column, then the cap at the outlet of the column. If caps are removed in reverse order, the column may run dry. Use a buffer without EDTA for metalloproteins
  9. Centrifuge the cleared lysates at 14,000 rpm for 5 min under 4°C. Insoluble aggregates formed during storage have to be removed to prevent clogging.
  10. Add supernatant of cleared lysates to the column. Volume of lysates should be in range 0.5 to 10 CVs. Extracts of large volume with recombinant proteins at low concentration may lead to the reduced yield.
  11. Wash the column 5 times with 1 CV Buffer W, after the cell extract has completely entered the column. Collect the eluate in fractions having a size of 1 CV. Apply 2 µl of the first washing fraction and 20 µl of each subsequent fraction to an analytical SDS-PAGE.
  12. Add 6 times 0.5 CVs Buffer E and collect the eluate in 0.5 CV fractions. 20 µl samples of each fraction can be used for SDS-PAGE analysis. Most of the purified Strep-tag® II fusion protein usually elutes in the 2nd to 5th fraction. Desthiobiotin and EDTA can be removed, if necessary, via dialysis or gel chromatography.

Troubleshooting

If the proteins don't bind to Strep-tag column

Problem Suggestions
pH is not correct The pH should be > 7.0
Strep-tag is not accessible Fuse Strep-tag with the other proteins terminus; use other linkers.
Strep-tag has been degraded Check the strep tag is not associated with the portion of proteins which is processed. Avoid purification in discontinuous batch mode.
Strep-tag column is inactive Check activity with HABA. Add 2-3 nmol of avidin monomer per nmol of biotin

If the proteins are contaminated or have low purity

Problem Suggestions
Contaminants are short forms of tagged proteins Add protease inhibitors after cell lysis. Fuse strep tag with other protein terminus. Check for the presence of the internal translation initiation sites or premature termination sites.
Covalent link between contaminants and recombinant proteins via disulphide bond. All reducing agents to all buffers for cell lysis and chromatography.

Air Bubble in the column

Problem Suggestions
Development of bubbles in the column bed Keep on working in the cold room. Wash columns immediately with buffers at ambient temperature once the column is removed from cold.

References

1: www.iba-lifesciences.com/

2: Schmidt, TGM and Skerra, A, 2007: NATURE PROTOCOLS 2, 1528-1535. The Streptag system for one-step purification and high-affinity detection or capturing of proteins.

3: Skerra A, Schmidt TGM, 2000: Meth. Enzymol. 326: 271-304. Use of the Strep-tag and streptavidin for recombinant protein purification and detection.

SDS-PAGE Protocol

Equipment

  • Erlenmeyer or plastic tubes
  • Hypodermic needle attached to the syringe
  • Pasteur pipette
  • Squirt Bottle
  • Micropipette equipped with gel loading tips
  • Vertical electrophoresis apparatus (Invitrogen)

Reagent

Buffer/Solution Contents
40% Acrylamide (37.5:1)
  • Acrylamide 116.8 g
  • N,N’-Methylene bisacrylamide 3.2 g
  • DDI H2O to 300 mL
  • Filter and store in a dark bottle at 4°C
30% Ammonium Persulfate
  • Ammonium Persulfate 1.5 g
  • DDI H2O 5 ml
  • Store at 4°C
RG Buffer--1.5 M
Tris•Cl, pH 8.8
  • DDI H2O 300 mL
  • Tris–free base 90.75 g
  • Conc. HCl 8 mL
SG Buffer--1.0 M
Tris•Cl, pH 6.8
  • DDI H2O 300 mL
  • Tris–free base 60.54 g
  • Conc. HCl 36 mL
4x SDS-PAGE Sample Buffer
  • 125 mM Tris•HCl, pH 6.8, 1 M, 5 mL
  • 20% Glycerol 8 mL
  • 4% SDS 20% 8 mL
  • 10% ß-Mercaptoethanol 4 mL
  • 0.5 mg/mL Bromophenol Blue 20 mg
  • DDI H2O 15 mL
10x SDS-PAGE Running Buffer
  • 30.3 g Tris base
  • 144.0 g Glycine
  • 10.0 g SDS
  • Fill upto 1000 mL with deionized water
Destain Solution
  • Ethanol 1200 mL
  • Glacial Acetic Acid 400 mL
  • DDI H2O 2.4 L

Casting the Gel

  1. Assemble glass plates and spacers in gel casting apparatus. See BioRad instruction manual.
  2. Mix the components for the resolving gel as described in the Mini-Protean II protocol.
  3. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the shorter plate.
  4. Place a layer of DDI H2O over the top of the resolving gel to prevent meniscus formation in the resolving gel.
  5. Allow resolving gel to stand 30 min at room temperature.
  6. Drain the DDI H2O from top of the resolving gel. Rinse with DDI H2O, drain, and wick any remaining DDI H2O away with a Kimwipe.
  7. Mix components for stacking gel.
  8. Pour stacking gel solution into gel plates (on top of running gel), so that gel plates are filled. Insert comb to the top of the spacers.
  9. Allow gel to stand for at least 1 hr at room temperature, or overnight at 4°C (wrapped in saran wrap).

Cell Samples

  1. Harvest 100 μL of cells at O.D. > 0.6. Decant the supernatant media.
  2. Resuspend cells in 20 μL of 2x sample buffer.
  3. Incubate tubes in boiling water for 5 min.
  4. Centrifuge at 12,000 x g for 30 s.

Solution Samples

  1. Place a volume of protein solution (or 1 μL of standard) into a μ fuge tube, such that there is 5-10 μg of protein in the solution.
  2. Add an equal volume of 2x sample buffer (or 10 μL for standards).
  3. Incubate tubes in boiling water for 5 min.
  4. Centrifuge at 12,000 x g for 30 s.

Running the Gel

  1. Remove comb and assemble cast gel into Mini-Protean II apparatus.
  2. Add freshly prepared 1x running buffer (300 mL) to both chambers of the apparatus.
  3. Load the prepared samples into the wells of the gel.
  4. Run the gel at 100 V until the dye front migrates into the running gel (~15 min), and increase to 200 V until the dye front reaches the bottom of the gel (~45 min).

Staining and Destaining the Gel

  1. Remove the run gel from the apparatus and remove the spacers and glass plates. Place the gel into a small tray.
  2. Add ~20 mL staining solution and stain for > 30 min with gentle shaking.
  3. Pour off and save the stain.
  4. Add ~5 mL destain solution and destain for ~1 min with gentle shaking.
  5. Pour off and discard the destain solution. Add ~30 mL of destain solution.
  6. Distain with gentle shaking until the gel is visibly destained (> 2 hr).
  7. Pour off and discard the destain solution.
  8. Rinse with DDI H2O. Add ~30 mL DDI H2O and rinse for 5 min with gentle shaking.
  9. Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper below the gel and a piece of Seran wrap over the gel.

% Acrylamide in running gel

Single Percentage Separation size range (kDa)
5% 100-250
7.5% 40-200
10% 30-150
12% 20-120
15% 10-100
18% 6-50
Gradient Separation size range (kDa)
4–15% 20–250
4–20% 10–200
10–20% 10–100
8–16% 6–70

References

1: The BioRad Readygel Manual.

2: Laemmli, U.K. (1970) Nature 227, 680-685.

3: Adapted from Laemmli, U.K. (1970) Nature 227, 680-685.

Ni-NTA Protein Binding Assay

Outline of Experimental Procedure

  • Membrane dialysis of His-tagged parasite protein to remove Imidazole used for purification of the protein against 150 mM KCl + 20 mM Hepes buffer
  • ↓
  • Verify thermal stability of cyclotides (Protocol 1)
  • ↓
  • Perform SDS PAGE to check the purity of proteins of interest
  • ↓
  • Measure the concentration of the host protein, parasite protein and cyclotide protein via bicinchoninic acid (BCA) assay (Protocol 2)
  • ↓
  • Concentrating protein solutions (if necessary) using centricon membrane filters of 10MWCO for storage
  • ↓
  • Resuspend the proteins in buffer solution and confirm the concentrations via BCA assay Buffer of 150 mM KCl + 20 mM Hepes
  • ↓
  • Wash NI-NTA functionalized agarose beads in a buffer and incubate with parasite protein in 96 well plate. Add host protein (control) or both host protein and cyclotide (test) and allow for binding (Protocol 3)
  • ↓
  • Directly or Indirectly measure cyclotide and host protein concentrations to check the effect of presence of cyclotide on host and parasite protein binding (Protocol 4)

Protocol 1

  • Add equal volumes of 1 μM BSA, 1 μM taq polymerase and 1 μM cyclotide into two eppendorf tubes.
  • Heat the first tube in a water bath (90°C for 10 mins).
  • Centrifuge contents of both tubes (30 mins at 20,000 g). Separate the supernatant from the pellet. Run supernatants and pellets of both tubes on SDS-PAGE compared to control lanes of taq polymerase, BSA and cyclotide individually. Presence of cyclotide in the supernatant after heat-denaturation is conclusive proof of the thermostability of the cyclotide.

Protocol 2: Bicinchoninic Acid Assay[1]

Reagent A: sodium bicinchoninate (1.0 g), Na2CO3 (2.0 g), sodium tartrate (dihydrate) (0.16 g), NaOH (0.4 g), NaHCO3 (0.95 g), made up to 100 mL. If necessary, adjust the pH to 11.25 with NaHCO3 or NaOH

Reagent B: CuSO4 5H2O (0.4 g) in 10 mL of water (see Note 1).

Standard working reagent (SWR): Mix 100 vol of regent A with 2 vol of Reagent B.

  • To a 100 μL aqueous sample containing 10–100 μg protein, add 2 mL of SWR. Incubate at 60°C for 30 min. The sensitivity of the assay can be increased by incubating the samples longer. Alternatively, if the color is becoming too dark, heating can be stopped earlier. Take care to treat standard samples similarly.
  • Cool the sample to room temperature, then measure the absorbance at 562 nm.
  • Standardization: A calibration curve can be constructed using dilutions of a stock 1 mg/mL solution of bovine serum albumin (BSA)

Protocol 3

  • Wash 20 µL (bed volume of a 1:1 slurry) Ni2+-NTA functionalized agarose beads with water to get rid of the ethanol in the slurry and then equilibrate beads with 150 mM KCl + 20 mM Hepes (HKS) Buffer
  • Add 500 µL of 1 µM of the parasite protein in HKS buffer containing the His tag to each well in the plate with the beads and incubate for 30 minutes at 37°C. Spin the beads after incubation and discard the supernatant containing excess unbound protein.
  • Add 500 µL of HKS Buffer to the beads. Mix well and centrifuge again on a table top centrifuge for 1 min. Discard the supernatant. Do the same wash three times.
  • For the host + parasite sample:
    • Add 500 µL of 1 µM host protein with the StrepII tag to each well in the plate and incubate at 37°C for 60 minutes.
    • Centrifuge, discard the supernatant, and resuspend the beads in buffer after washing (in buffer) 3 times.
    • Add 20 µL of 2x Laemmli’s buffer to the beads.
    • Load the samples on a SDS PAGE to verify the proteins (Control)
    • Obtain concentration of host protein (see Protocol 4)
  • For the host + parasite +cyclotide sample (Method 1):
    • Add between 100 nM to 10µM cyclotides (gradation in concentration)in 500 µL volume to each well in the plate and incubate at 37°C for 60 minutes.
    • Centrifuge, discard the supernatant, and resuspend the pellet in buffer after washing (3x times) to get rid of unbound cyclotide.
    • Add 500 µL of 1 µM of the host protein with the StrepII tag to each well in the plate and spin at 37°C for 60 minutes.
    • Centrifuge, discard the supernatant, and resuspend the pellet in buffer after washing (in buffer) 3 times.
    • Add 20 µL of 2x Laemmli’s buffer to the beads.
    • Perform SDS PAGE for qualitative estimation of inhibition of binding in presence of cyclotide.
    • Do BCA assay of the proteins bound on the beads to get a quantitative estimate of inhibition of binding (see Protocol 4)
  • For the host + parasite +cyclotide sample (Method 2):
    • Mix the amount of cyclotide showing most inhibition of binding determined by method 1 along with 500 µL of 1 µM of the host protein to each well of parasite protein-functionalized beads and incubate at 37°C for 60 minutes.
    • Centrifuge, discard the supernatant, and resuspend the pellet in the buffer after washing to get rid of excess unbound cyclotide and host protein in solution.
    • Add 20 µL of 2x Laemmli’s buffer to the beads.
    • Perform SDS PAGE to check if the amount of host protein is lesser than the control (parasite protein on beads+host protein)
    • Obtain concentration of host protein (see Protocol 4)

Protocol 4

  • Direct measurement of bound protein:
    • Set up two identical reactions with the parasite protein
    • Add 500 µL 1 µM of the parasite protein with the His tag to both reaction setups and incubate at 37°C for 30 minutes.
    • Centrifuge, discard the supernatant, and resuspend the pellet in buffer after washing in both setups.
    • Add 1% SDS to the beads to denature the protein and get it in solution. Do a BCA assay of this to get an estimate of the amount of parasite protein bound on 20 µL of beads under equilibrium conditions. Let this concentration be p.
    • For host + parasite protein sample:
      • After allowing the host protein to bind and centrifuging and obtaining the pellet (see Protocol 3), obtain the protein remaining bound to the beads by adding 1% SDS to the beads. Measure concentration of this total protein via BCA. Let this concentration be x.
      • Concentration of host protein bound h = x - p
    • For host + parasite + cyclotide protein sample:
      • After allowing the host protein and cyclotide to bind and centrifuging and obtaining the pellet (see Protocol 3), obtain the total protein bound to the beads by adding EDTA to a final concentration of 1 mM. Spin to get rid of the beads.
      • Take the supernatant and heat-denature the total protein by subjecting it to a water-bath heated at 90°C for 10 mins. Spin the sample at 20,000g for 30 mins to separate supernatant and pellet. The supernatant will contain the cyclotide. Pellet will contain host protein, parasite protein and beads.
      • Perform BCA on the supernatant. Let this concentration be c.
      • Concentration of cyclotide = c
      • Obtain all the protein in the pellet and perform BCA. Let this concentration be x’.
      • Concentration of host protein h = x’ - p
  • Indirect measurement of bound protein:
    • Allow parasite protein to bind to the agarose beads according to protocol 3. Let initial concentration of parasite protein be pi. Centrifuge and perform BCA on the supernatant. Let this concentration be pf.
    • Concentration of parasite protein bound p = pf - pi
    • For host + parasite protein sample:
      • Perform a BCA on host protein before adding it to the reaction setup. Let this concentration be hi.
      • After allowing the host protein to bind and centrifuging and obtaining the pellet (see Protocol 3), perform a BCA of the supernatant. Let this concentration be hf.
      • Concentration of host protein bound = hf - hi.
    • For host + parasite + cyclotide protein sample:
      • Perform a BCA on host protein before adding it to the reaction setup. Let this concentration be hi.
      • Perform a BCA on cyclotide before adding it to the reaction setup. Let this concentration be ci.
      • After allowing the host protein and cyclotide to bind and centrifuging and obtaining the supernatant (see Protocol 3), thermally precipitate it. Supernatant will contain unbound cyclotides. Pellet will contain unbound host protein.
      • Perform BCA on the supernatant. Let this concentration be cf.
      • Concentration of cyclotide = ci - cf
      • Obtain all the protein in the pellet and perform BCA. Let this concentration be hf.
      • Concentration of host protein h = hi - hf

References

1: Walker, J. M. (2009). The Protein Protocols Handbook (Springer Protocols Handbooks) (3rd ed.). New York City, NY: Humana Press.

IC50 Assay

SYBR Green I DNA Fluorometry

Materials and Reagents

  • P. falciparum culture
  • Sterile 96-well plate
  • Varioskan Flash
  • SYBR Green I DNA staining dye
    [NB: SYBR Green I is light-sensitive; keep it in the dark as much as possible]
Buffer Concentration of Ingredients
Lysis buffer
  • 100 mM Tris-HCl
  • pH 7.5
  • 25 mM EDTA
  • 0.5% Triton X-100
  • 0.05% saponin in PBS

Protocol

  1. Take 100 µL of RPMI in the wells of 96 well-plate.
  2. Choose a concentration range for the experiment.
  3. Prepare a 200 µM stock of the drug in RPMI.
  4. Perform serial dilution (2-fold)
    1. Take 100 µL of the stock solution and transfer it into a well containing 100 µL RPMI (the drug concentration of the solution is now reduced to 100 µM)
    2. Take 100 µL of solution from well 1 and transfer it into well 2 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 50 µM)
    3. Take 100 µL of solution from well 2 and transfer it into well 3 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 25 µM)
    4. Take 100 µL of solution from well 3 and transfer it into well 4 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 12.5 µM)
    5. Take 100 µL of solution from well 4 and transfer it into well 5 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 6.25 µM)
    6. Take 100 µL of solution from well 5 and transfer it into well 6 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 3.125 µM)
    7. Take 100 µL of solution from well 6 and transfer it into well 7 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 1.5625 µM)
    8. Take 100 µL of solution from well 7 and transfer it into well 8 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 0.78125 µM)
  5. Take 100 µL of the culture (RBC + RPMI) with 4% hematocrit value and 2% parasitemia and transfer it in well 1 to 8.
    [NOTE: Now the hematocrit value of the solution is reduced to 2% but the parasitemia remains the same. The drug concentration is also reduced by half.]
  6. Controls
    1. Blank (background signal control)
      1. Take 100 µL of RPMI in well 9
      2. Add 100 µL of culture (RBC + RPMI) with 4% hematocrit and no parasites
    2. RPMI control (zero drug control)
      1. Take 100 µL of RPMI in well 10
      2. Add 100 µL of culture (RBC + RPMI) with 4% hematocrit value and 2% parasitemia
    3. DMSO control (to check the effect of solvent)
      1. Take 100 µL of DMSO in well 11
      2. Add 100 µL of culture (RBC + RPMI) with 4% hematocrit value and 2% parasitemia
  7. The plate is incubated for 48 hours at 37°C under the gas conditions of 5% CO2 and 95% N2
  8. After 48 hours transfer the plate to -80°C to freeze.
  9. Prepare a 10X stock of SYBR Green 1 + Lysis Buffer (volume = 2500 µL)
  10. Thaw the plate at room temperature (this freezing and thawing will cause cell lysis)
  11. Add 22.5 µL of 10X SYBR Green 1 + Lysis buffer to each well
  12. Mix and incubate for 45 minutes at room temperature in dark
  13. Take fluorometer readings in Thermo Fisher VarioskanFlash
    1. Select the lane
    2. Select fluorometry based assessment
    3. Set up mode to:
      Excitation 495 nm
      Emission 520 nm
    4. Select type of plate i.e. Flat Bottom/Conical/Round Bottom
    5. Record the fluorescence values
  14. Import data into this excel sheet.
    Download Excel Spreadsheet

Analysis

  1. We can calculate the IC50 values by using an online tool, IC50 calculator.[1]

    The tool uses a four parameter logistic regression model to calculate the value. This model typically resolves as a sigmoid function, or "S"-shaped curve.

    All you have to do is input the concentration and readout values obtained from the excel sheet.

  2. We have also written R code which you can use to calculate the IC50 value. For the code to work you have to save the concentration and readout values obtained in the Excel sheet in two columns in a CSV file.[2]

    Download R code file

Troubleshooting

  1. To check if the proposed drug acts as an inhibitor we first select a broad drug concentration range and if the IC50 value is obtained then the experiment is performed again with a narrow drug concentration range to get a more accurate value.
  2. After the results are obtained if the growth in zero drug concentration is less than the growth in any well with some drug concentration then it is because of handling error. We have either added more cells in a well or more dye in a well. To avoid this the experiment is done in triplets and average is taken.
  3. We perform RPMI and DMSO control to check if the drug solvent is responsible for any inhibition effect or not. DMSO is the drug solvent. If the fluorometer readings of DMSO control and RPMI control are similar then that means solvent is not contributing to the inhibition effect.
  4. We perform a blank control with no parasites (no DNA in sample) so that we can deduct the background noise from all readings.

References

1: "IC50 Calculator | AAT Bioquest." www.aatbio.com/tools/ic50-calculator. Accessed 26 Oct. 2020.

2: "Bioassay analysis using R - Journal of Statistical Software." 23 Jan. 2005, www.jstatsoft.org/v12/i05/paper. Accessed 14 Oct. 2020.

3: SYBR Green I DNA Fluorometry, sites.psu.edu/llinaslab/files/2020/05/SYBR_Green_protocol.pdf.

RBC Invasion Assay

Materials Required

  • Chemicals
    • RPMI-1640 media
    • HEPES
    • Sodium bicarbonate
    • Hypoxanthine
    • Albumax II
    • α-2-3,6,8–Vibrio cholera neuraminidase
    • Trypsin
    • Chymotrypsin
    • Phosphate buffered saline(PBS)
    • Bovine serum albumin (BSA)
    • Sodium azide
    • Sorbitol
    • SYBR Green I dye
  • Others
    • 96-well microtiter plate

Protocol

Parasite Culture

  • Solution 1: 4% hematocrit in RPMI-1640 media supplemented with 25 mM HEPES, 0.21% sodium bicarbonate, 50 mg/L hypoxanthine, and 0.5% Albumax II
  • Solution 2: α-2-3,6,8– Vibrio cholera neuraminidase (66.7 mU/mL, Calbiochem), trypsin (1 mg/ mL, Sigma), and chymotrypsin (1 mg/mL, Worthington)
  1. Treat the P.falciparum asexual stages maintained in vitro in human O+ erythrocytes with solution 1.
  2. Perform invasion assays by mixing enzyme-treated infected donor cells with equivalent cell number of RPMI treated erythrocyte control cells, or enzyme-treated erythrocyte negative control cells.
  3. Treat ring stage infected O+ erythrocytes with solution 2 to prevent reinvasion.
  4. Plate parasite duplicates in complete RPMI media at a final parasitemia of between 0.45 and 1% at 2% hematocrit.
  5. Following reinvasion, about 48 hours after plating, assess parasitemia by flow cytometry as detailed below.

Flow Cytometry

  • Solution 3: 1 x phosphate buffered saline (PBS) + 0.5% bovine serum albumin (BSA) + 0.02% sodium azide
  1. Culture two-hundred microliters of sorbitol-synchronized, ring-stage parasites in a 96-well microtiter plate at 1% parasitemia.
  2. Plate samples in triplicate and incubate at 37℃ until reinvasion.
  3. Harvest cultures through centrifugation (1200 rpm, 5 minutes) and wash it twice in 100 μL of solution 3.
  4. Incubate cells with 75 μL of 1:1000 SYBR Green I (Molecular Probes) for 20 min at 25℃.
  5. Wash the cells in solution 3 and resuspend it in PBS.
  6. Collect flow cytometry data using FACS ARIA. Carry out initial gating with unstained, uninfected erythrocytes to account for erythrocyte autofluorescence. Analyse the data with FlowJo 8.8.6 software (Tree Star).

Precautions

The accuracy with which flow cytometry resolves multiply-infected erythrocyte peaks needs the culture to be at the ring-stage. As the culture shifts from late rings to early trophozoites the parasite starts to replicate its DNA. The flow cytometer is unable to distinguish between the fluorescence emitted by a multiple infected erythrocyte having more than one, ring stage parasites versus one infected by a single, early trophozoite. The requirement for ring-stage parasites can easily be met for in vitro laboratory-adapted parasites cultured by synchronization by sorbitol lysis.

Troubleshooting

  1. In step 3, if treatment of infected erythrocytes with solution 2 is very harsh because of the α-2-3,6,8– Vibrio cholera neuraminidase and trypsin and chymotrypsin (all three chemicals block reinvasion completely) , lower concentrations can be used and the relative effectiveness of different peptide inhibitors can be studied.
  2. If FACS does not give promising results because of lack of standardization then fluorometry based analysis can be performed using a spectrophotometer, which will be speedier as well.
  3. Harsh sorbitol treatment can affect reinvasion. To troubleshoot this, fresh parasite cultures can be revived to grow in a synchronized manner for the initial cycles. Sorbitol treatment should be avoided as much as possible.

References

1: Bei, A. K., DeSimone, T. M., Badiane, A. S., Ahouidi, A. D., Dieye, T., Ndiaye, D., … Duraisingh, M. T. (2010). A flow cytometry-based assay for measuring invasion of red blood cells byPlasmodium falciparum. American Journal of Hematology, 85(4), 234–237. doi.org/10.1002/ajh.21642

2: Jacobberger JW, Horan PK, Hare JD: Analysis of malaria parasite-infected blood by flow cytometry. Cytometry 1983, 4:228–237.
www.sciencedirect.com/science/article/abs/pii/016511619390140U

PCR Protocol

Cloning

Enzyme: Pfu DNA polymerase

Template: Synthesized Gene

Steps

  1. Gradient Amplification to determine optimal annealing temperature

    Gradient PCR is a modification to a standard PCR procedure that allows to optimize an annealing temperature in order to increase the specificity of the amplification process. It is usually conducted in a thermal cycler in which different annealing temperatures can be set in different parts of the block, maintaining constant denaturing and elongation temperatures. Thus, after running a gel with PCR products and comparing the pattern of bands, it is possible to empirically determine the optimal annealing temperature for a given set of primers. Annealing temperatures between 55°C to 65°C are ideal for PCR reaction. Deviation of annealing temperature above or below this range can cause non-specific bindings or reaction failure.

  2. Gene amplification at annealing temp determined by gradient PCR
Name 50 μL reaction 20 μL reaction
Molecular Grade Water Make up to 50 Make up to 20
10X ThermoPol®️ Buffer 10 μL 4 μL
dNTPs 2 μL 0.8 μL
Forward primer X μL 2X/5 μL
Phusion Polymerase 1 μL 0.4 μL
Reverse primer X μL 2X/5 μL
DNA X μL 2X/5 μL
Step Temp. Time
Hold 1 Initial denaturation 95°C
Cycling x30 Denaturing 98°C 10 s
Annealing X°C 30 s
Extension 72°C 30 s/kb
Hold 1 Final extension 72°C 10 mins

Tip: Use 15-30 s/kb for extension. Do not exceed 1 min/kb.

Clone Confirmation

Enzyme: Pfu DNA polymerase

Template: pET-28a(+) vector

Name 50 μL reaction 20 μL reaction
Molecular Grade Water Make up to 50 Make up to 20
10X ThermoPol®️ Buffer 10 μL 4 μL
dNTPs 2 μL 0.8 μL
Forward primer (M13) X μL 2X/5 μL
Pfu Polymerase 1 μL 0.4 μL
Reverse primer (M13) X μL 2X/5 μL
DNA X μL 2X/5 μL
Step Temp. Time
Hold 1 Initial denaturation 95°C
Cycling x30 Denaturing 98°C 10 s
Annealing 50°C 30 s
Elongation 72°C 30 s/kb
Hold 1 Final extension 72°C 10 mins

Troubleshooting

→ No PCR product is observed
Possible Cause Solution
A PCR component is missing or degraded A positive control should always be run to insure components are functioning. A checklist is also recommended when assembling reactions.
Too few cycles were performed Increase the number of cycles (3-5 additional cycles at a time).
The annealing temperature is too high Decrease the annealing temperature in 2-4°C increments
The primers are not designed optimally Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primer to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%.
There is not enough template After increasing the number of cycles has shown no success, repeat the reaction with a higher concentration of template.
The template is of poor quality Evaluate the template integrity by gel electrophoresis. It may be necessary to repurify the template using methods that minimize shearing and nicking.
The denaturation temperature is too high or too low Optimize the denaturation temperature by increasing or decreasing the temperature in 1°C increments.
The denaturation time is too long or too short Optimize the denaturation time by increasing or decreasing it in 10-second increments.
The extension time is too short Increase the extension time in 2-minute increments, especially for long templates.
The reaction does not have enough enzyme 1.0 µL (2.5 units) is sufficient for most applications. It is recommended that the cycling parameters be optimized before the enzyme concentration is increased. In rare cases, the yields can be improved by increasing the enzyme concentration. However, if the enzyme amount is above 2 µl (5 units), higher background levels may be seen.
Mg2+ levels are suboptimal This is unlikely if the 10X reaction buffer (with MgCl2) is used and the deoxynucleotides do not exceed a concentration of 0.6 mM each (as deoxynucleotide triphosphates can bind Mg2+). Typically, MgCl2 is optimized between 1 to 5 mM. Also, EDTA present in the sample at greater than 5 mM will reduce the effective concentration of magnesium.
Deoxynucleotide concentration is too low This is unlikely if the final concentration of each deoxynucleotide is 0.5 mM. This concentration of dNTPs is suitable for a wide range of applications. If the dNTPs are being prepared in the laboratory, be sure that the final concentration of each deoxynucleotide is 0.5 mM. If the concentration of dNTPs is increased, the Mg2+ concentration will need to be increased proportionately.
Target template is complex In most cases, inherently complex targets are due to unusually high GC content and/or secondary structure.
→ There are multiple or smeared products
Possible Cause Solution
The annealing temperature is too low Increase the annealing temperature in increments of 2-3°C.
The primers are not designed optimally Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primers to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primers with a GC content of 45-60%.
Touchdown PCR may be required “Touchdown” PCR significantly improves the specificity of many PCR reactions in various applications. Touchdown PCR involves using an annealing/extension temperature that is higher than the TM of the primers during the initial PCR cycles. The annealing/extension temperature is then reduced to the primer TM for the remaining PCR cycles. The change can be performed in a single step or in increments over several cycles.
Too many cycles were performed The nonspecific bands may be eliminated by reducing the number of cycles.
There is too much enzyme in the reaction mix 1 µL (2.5 units) is sufficient for most applications. However, this concentration may be too high for some applications. We recommend optimizing the cycling parameters first as described above, then if necessary incrementally reduce the enzyme concentration to determine the optimal concentration.
Magnesium concentration is too high The MgCl2 concentration should be optimized. Typically, the concentration of MgCl2 is optimal between 1 and 5 mM. If the concentration of the dNTPs is 0.5 mM, it is very unlikely that the magnesium concentration is too high.
The template concentration is too high Reduce the concentration of the template in the PCR reaction.
The template concentration is too low Add additional template in 50 ng increments for genomic DNA or 1-2 ng for viral DNA.
→ The yield of specific products is low
Possible Cause Solution
Too few cycles were performed Increase the cycle number in 3-5 cycle increments.
Extension times are too short Increase the extension times in 2-minute increments.
A co-solvent is required Add dimethyl sulfoxide up to a final concentration of 5%.
PCR priming opportunities may be low due to reaction conditions or primer design Modify the reaction conditions by increasing the denaturation temperature to 95°C, increase extension times in 2-minute increments, increase MgCl2 and dNTP concentrations, etc. Redesign PCR primers.

Reference

https://www.sigmaaldrich.com/technical-documents/protocols/biology/long-and-accurate-dna-amplification.html
Link

Site Directed Mutagenesis Protocol

Background

This protocol has been adapted from the Team Wageningen_UR 2014 which is based on the Stratagene Quikchange protocol. The primary reaction is illustrated below. Here the product of the reaction is never used as a template. This is a linear amplification technique, unlike standard PCR where an exponential amplification of the product is obtained.

Protocol

  • Oligo Designing

    The protocol in the QuikChange Manual is followed for oligo-design. It designs a primer with a Tm of at least 78°C and centers the mutation in the middle, which works well for single amino acid changes. An automated website to help do this is available at bioinformatics.org/primerx. According to the Stratagene protocol, the oligos need to be PAGE purified.

  • Solution
    • 0.5 µL 2.5 pmoles/µL forward primer
    • 0.5 µL 2.5 pmoles/µL reverse primer
    • 0.25 µL 40 mM dNTP mix (10 mM each)
    • 1 µL 2 ng/µL Template DNA
    • 1.25 µL 10x PfuUltra Buffer (contains Mg2+)
    • 0.25 µL PfuUltra Hotstart (Stratagene)
    • 8.75 µL sterile H2O
  • PCR Program
    • Run for 5 min at 95°C
    • Repeat 18x
      1. 50s at 95°C
      2. 50s at 60°C
      3. 1 min + 1 min/1 kb template at 68°C
    • Run again for 7 min at 68°C
  • Run 2.5 µL of the reaction on a gel, there should be a band corresponding to your product.
  • Add 0.25 µL of DpnI to the reaction. Incubate at 37°C for 1 hr.
  • Transform the final reaction into competent cells.
  • Pick a colony, isolate its plasmid DNA and sequence it to check for the mutation and any PCR introduced errors.

Precautions and Troubleshooting

  1. The digestion with Dpnl is crucial as it only cuts at methylated sites. Since the transformation efficiency of the circular template plasmid is better than the linear PCR product, without the DpnI digest, a large number of colonies would be parental. It implies that the template plasmid cannot come from a methylation deficient strain (e.g., JM101).
  2. As the PCR reaction goes around the entire plasmid, we need to minimize the chances of introducing unwanted mutations in your gene and the backbone. So a high fidelity polymerase is also crucial.
  3. The concentration of primer versus template decides whether primer-primer or template-primer interaction dominates. If a reaction fails and if a strong primer dimer band is seen, it means that primer-primer annealing is favored over primer-template annealing. To troubleshoot this, the following solutions can be implemented: decreasing the primer concentration, increasing the template concentration, decreasing the annealing temperature, or redesigning primers.
  4. In case no product is seen, repeating the protocol with 5% DMSO in the solution mix can help.DMSO disrupts base pairing, facilitating strand separation in GC rich regions of DNA, and reducing the propensity of the DNA to form secondary structure.

References

1: L Zheng, U Baumann, and JL Reymond. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucl. Acids Res., 32:e115, 2004.

2: iGEM Team Wageningen 2014, Standard Protocol for Mutagenesis


Team IISER Pune India