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Experiment

Competent Cell Preparation

Introduction

Preparing competent cells of E. coli strains BL21, DH5 alpha, and TOP10 involves a specific set of steps. Competent cells are vital for efficient transformation and DNA uptake during experiments.

Materials

Procedure

  1. Pick a single colony of cell and inoculate in 10 ml of LB.
  2. Grow it overnight at 37°C with shaking.
  3. Add 1 ml of overnight grown culture to 100 ml of pre-warmed LB broth in a 500 ml flask.
  4. Shake and grow at 37°C until the OD reaches 0.5.
  5. Cool the culture on ice for 5 min and then transfer it to a round-bottom centrifuge tube (Falcon).
  6. Collect the cells by centrifugation (5 min, 4000g) at 4°C.
  7. Discard the supernatant and keep the cells on ice.
  8. Resuspend the cells in cold TFB-1 buffer (30 ml) and incubate on ice for 90 min.
  9. Collect the cells by centrifugation (5 min, 4000g, 4°C).
  10. Discard the supernatant and keep the cells on ice.
  11. Resuspend the cells in 4 ml of ice-cold TFB-2.
  12. Prepare 50 to 100 ml aliquots in 1.5 ml centrifuge tubes.
  13. Store the tubes in -80°C.

Caution

  • All steps must be performed on ice.
  • The lab hood should be bathed in UV light for more than 30 minutes before starting the procedure.

Transformation

Introduction

Transfer of miniprep plasmids or ligation mixture into Escherichia coli KRX or BL21.

Materials

  • Ice
  • Competent cells
  • Ligation mixture or miniprep plasmids
  • SOC Medium
  • Hot water bath
  • Incubator
  • Timer
  • Agar plates
  • Cell spreader
  • Bunsen burner
  • Laminar air flow hood

Procedure

  1. Take out the competent cells (100μl) from -80°C storage and place in ice.
  2. Add ligation mixture (10μl) or miniprep Plasmids (1 or 2 μl) to the 100μl competent cells by pipetting.
  3. Close the tube and mix them by gently tapping on the side of the tube.
  4. Keep the tube in ice for 30 mins.
  5. Set the hot water bath to 42°C and keep the SOC media in the incubator at 37°C.
  6. Heat shock the tube for 45 seconds at 42°C, then place it back in ice for 5 mins.
  7. Add SOC media to make up the volume to 1000μl and incubate it for 1.5 hours with shaking at 37°C.
  8. Spread 100 μl on an agar plate and incubate at 37°C for 12-16 hours.

Caution

The LB can be used as a media, but SOC is more favorable for bacterial growth and efficient colonies.

Cloning Method



For Plasmid Extraction: We followed the Qiagen Protocol.


Notes before starting:


  1. 1. Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
  2. 2. Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C.
  3. 3. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
  4. 4. All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.

Procedure:


  1. 1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
  2. 2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. 3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  4. 4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. 5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. 6.Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
  7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
  8. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
  9. 7. Transfer the QIAprep 2.0 spin column to the collection tube.
  10. 8. Centrifuge for 1 min to rem/7ove residual wash buffer.
  11. 9.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
  12. 10. If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

To get a better yield, we strictly followed the following steps:


  1. 1. After adding P2 buffer invert the tube vigorously and keep the tube still only for 1.5 min. (not more than that).
  2. 2. Adding the N3 buffer again invert the tube 4-6 times and centrifuge it for 20 min at 13,000 rpm.
  3. 3. Now carefully with the help of 20-200 uL pipette pull out the transparent part without disturbing the white part.
  4. 4. Keep the spin column for 2 min in the rack and then spin it for 30 sec .
  5. 5. Now have to add PB and keep the tube undisturbed for 5 min and again spin it for 2 min.
  6. 6. Adding the PE buffer and spinning it three times.
  7. 7. Now during the Elution we need to first add an elution buffer (20 ul) and keep the tube for 15 min and spin it for 2 min and we have to repeat the same process again.

For Gel Extraction: We followed the Qiagen Protocol.


Notes before starting:


  1. 1. This protocol is for the purification of up to 10 μg DNA (70 bp to 10 kb).
  2. 2. The yellow color of Buffer QG indicates a pH ≤+7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.
  3. 3. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
  4. 4. Isopropanol (100%) and a heating block or water bath at 50°C are required.
  5. 5. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

Procedure


  1. 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. 2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
  3. 3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
  4. 4. Add 1 gel volume isopropanol to the sample and mix.
  5. 5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 μl, load and spin/apply vacuum again.
  6. 6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
  7. 7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt- ended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
  8. 8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  9. 9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let thecolumn stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
  10. 10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

To get a better yield, we strictly followed the following steps:


  1. 1. To get a better yield for the lengths of less than 600 bp we need to run the gel in presence of TBE buffer in 2.8 % gel media, which ultimately during extraction needs more than 5.5 % of QG buffer.
  2. 2. Also need to use more than 1.5 % of the weight of isopropanol to minimize co precipitation of salt that interferes with downstream applications.
  3. 3. Need to do centrifugation for 5 min at 13,000 rpm and instead of throwing the flow through spin it again for 1 min to extract the ultimate product from the sample.
  4. 4. During the usage of the wash buffer spin it three times for 60 sec to get rid of all the ethanol from the solution.
  5. 5. Do the elution with 55 degree centigrade hot NFW to reduce the amount of salt.

Gel Electrophoresis

Introduction

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.

Materials

Procedure

  1. To make 1% Agarose gel, mix 1g of Agarose in 100ml of 1X TAE.
  2. To dissolve, microwave it for 2-3 mins until it completely dissolves with occasional swirling. Remember not to over-boil as some amount can get evaporated and spilled.
  3. Let it cool to 50°C.
  4. Add EtBr to a final concentration of 0.2 - 0.5 μg/ml.
  5. Cast the gel in the gel tray and place the comb carefully.
  6. Keep the gel at room temperature for 20-25 mins (till it solidifies properly).
  7. Add loading dye to each sample.
  8. Fill the gel box with 1X TAE buffer, then place the solidified agarose gel in the gel box . Remember to check if the gel is fully covered with the buffer, if not so then add some more buffer!
  9. Load the ladder in the first lane. Then carefully load samples.
  10. Run the gel at 80-120 Volt until the dye line is approximately 75-80% of the way down the gel. Should take 1-1.5 hrs approximately.
  11. Remove the Gel carefully and then visualize it using UV.
  12. Now add 1 ml of autoclave water to the 1 mg of the etbr and vortex it until the complete ppt has gone .

Caution

  • For EtBr Prep - Wear goggles and maintain a safe distance.

Table: Agarose Gel % and Length of DNA to resolve.

Agarose Gel % Length of DNA to resolve
0.5 1000 - 30000 bp
0.7 800 - 12000 bp
1.0 500 - 10000 bp
1.2 400 - 7000 bp
1.5 200 - 3000 bp
2.0 50 - 2000 bp

Polymerase Chain Reaction (PCR)

Amplification PCR

Introduction

PCR is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample, allowing us to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail.


Materials


Procedure

  1. 1. Prepare the PCR mix by adding the following reagents by placing the PCR tubes all on ice.

    2. Reagents Volume (μL)
    NEB Q5® High-Fidelity 2X Master Mix 25
    Forward Primer 2.5
    Reverse Primer 2.5
    Template DNA 3
    NFW 17
  2. 2. Rest of the protocol to be followed as provided by NEB (linked in references).

Gradient PCR

We have followed the same protocol as the NEB provided (linked in references).


Colony PCR

Introduction


Colony PCR is a method for rapidly screening colonies of bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.


Materials

Procedure

  1. 1. Fill PCR tubes (as many required) with 10μl of NFW in each tube.
  2. 2. The culture plates with colonies were marked accordingly.
  3. 3. One colony was picked with a sterile tip and was streaked at the marked position on the new plate.
  4. 4. Then put the tip in the PCR tube containing 10μl of NFW.
  5. 5. Take the same number of autoclaved PCR tubes as number of colonies picked and label accordingly.
  6. 6. Add the following reagents in the PCR tubes to make a reaction mixture of 25μl.

    7. Reagents Volume (μL)
    Go Taq Green DNA master mix 12.5
    VF2 1
    VR 1
    Template DNA 5
    NFW 5.5
  7. The only difference is that we have to set the denaturation temp at around 95 degrees centigrade for 10 min.

PCR Optimised for Amplification of Small Lengths of DNA

Introduction

In the very beginning of the reaction, we have to heat the lid and PCR machine up to 98 degrees Celsius and then from the ice directly have to put the tubes inside the PCR machine.

Then follow the regular rule of PCR.

This is basically optimized PCR protocol for amplification of the short lengths (100-400 bp).



HiFi Assembly

Introduction

NEBilder HiFi Assembly: To assemble the inserts and the backbone and check the induction.


Optimal Quantities

NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool, NEBiocalculator (linked in references).


pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)

50 ng of 5000 bp dsDNA is about 0.015 pmols

50 ng of 500 bp dsDNA is about 0.15 pmols


The mass of each fragment can be measured using the Nanodrop instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.


HiFi DNA Assembly Protocol

  1. 1. Set up the following reaction on ice as follows.

    2. Headings: Recommended Amount of Fragments Used for Assembly


    2–3 Fragment Assembly* 4-6 Fragment Assembly** NEBuilder Positive Control✝
    Recommended DNA Molar Ratio vector: insert = 1:2 vector: insert = 1:1
    Total Amount of Fragments 0.03–0.2 pmols* X μl 0.2–0.5 pmols** X μl 10 μl
    NEBuilder HiFi DNA Assembly Master Mix 10 μl 10 μl 10 μl
    Deionized H2O 10-X μl 10-X μl 0
    Total Volume 20 μl 20 μl 20 μl
  2. 2. Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.
  3. To achieve optimal assembly efficiency, design 20-30 bp overlap regions between each fragment with equimolar of all fragments (suggested: 0.05 pmol each).
  4. Control reagents are provided for 5 experiments.
  5. If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NE Builder HiFi DNA Assembly Master Mix.

Single Digestion

We recommend digesting 0.2-1.5 µg DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 µl. A typical restriction enzyme digestion protocol is below.

Protocol:

  1. Add the following reaction components in the order indicated:

    2. Component Volume (μL)
    Water, nuclease-free 16-16.5
    10X recommended buffer for restriction enzyme 2
    Substrate DNA 1 (~1 µg)
    Restriction enzyme 0.5-1 (5-10 u)
    Total 20
  2. 2. Mix gently and spin down briefly.
  3. 3. Incubate at the optimal reaction temperature for 1-16 hours.
  4. Note: The digestion reaction may be scaled either up or down. Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately.

References

  1. https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/8-DNA-ladder-tips.html
  2. https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492
  3. https://ww.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
  4. NEBiocalculato
  5. https://www.neb.com/en/protocols/2018/07/30/restriction-digest-protocol
  6. https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492