Protocols

Agarose gels and DNA Electroforesis protocol Bacterial growth model in the presence of sieved by-products protocol Golden Gate protocol Miniprep protocol Mix and Go Competent Cells protocol" Neb Turbo Competent (C2984H) transformation protocol PCR protocol Pomace treatment protocol

Agarose gels and DNA Electroforesis protocol

BASIS

Plasmid DNA electrophoresis is an efficient technique to analyse and detect cloned plasmids in bacteria. From an electric field, DNA molecules migrate from the negative to the positive pole according to their size. With the help of a molecular weight marker, it is also possible to analyse the size of the resulting bands in base pairs.

MATERIAL

REAGENTS

PROCEDURE

  1. Preparation of the agarose solution. The size of the gel required must be taken into account and depends accordingly on the number of lanes required. The quantities required according to the type of gel are given below:
    2. For 8 lanes or less (small gel) For more than 8 lanes (normal gel)
    Agarose 0.25 g 0.5 g
    TAE 1X 25 mL 50 mL
    Green Save (must be added in step 3) 1 uL 2 uL
  2. Heat TAE+agarose until completely dissolved, resulting in a clear, lump-free solution.
  3. The solution is allowed to cool sufficiently. After this time, Green Save is added in a variable volume according to the size of the gel (see table above). It is important to keep the Green Save at low temperatures. Mix gently.
  4. Prepare the agarose solution cuvette. Choose the size corresponding to the gel and include the necessary comb to mark the wells. Add the agarose solution on top.
  5. Allow the solution to cool until completely solidified.
  6. Transfer the cuvette with the gel to the gel box. Add TAE 1X.
  7. Load the samples. It is necessary to add loading buffer if the samples are not enzymatic digests. If we are in a digestion it will also be necessary to add a molecular weight marker.
  8. Close the electrophoresis cuvette and connect it to the power supply, taking care to position the sockets correctly. Turn on the power supply and set the time and voltage required.

Bacterial growth model in the presence of sieved by-products protocol

BASIS

We have performed a bacterial growth test of BL21 E. coli bacteria with different concentrations of the extracted sieved wine by-products that we plan to use to boost the bioproduction of resveratrol once the final construction has been obtained.

MATERIAL AND REAGENTS

PROCEDURE

  1. Extract preparation

    2.5 grams of fermented extract were weighed and 10 ml of 96º Ethanol was added. It was transferred to a mortar and pestle and the residue was crushed. The supernatant was centrifuged and then filtered for sterility. In this way, we obtained an extract corresponding to 0.25 grams/ml ethanol. By adding 200 ul to 1 ml of culture medium, we would be adding 0.05 grams, achieving 5 mgr/100 ml, i.e. 5% w/v. By adding 40 ul, we achieve 1% and by adding 4 ul we achieve 0.1%.

  2. Cultures

    Each well has 1 ml of LB. We can prepare 20 ml of LB + 20 ul of Spectinomycin and add to each well. Then add the necessary amount of extracts and then inoculate with 10 ul of coli culture (except blanks).

    Blank LB Blank 0.1% Blank 1% Blank 5% Blank EtOH 200 µL Blank EtOH 40 µL
    Control 1 0.1% 1 1% 1 5% 1 1 1
    Control 2 0.1% 1 1% 2/td> 5% 2 2 2
    Control 3 0.1% 1% 3 5% 3 3 3
  3. OD measurement

    Bacterial optical density can be measured by spectrophotometry over a period of 15 to 24 hours at 30 min intervals. Stirring during the growth period should be constant.

Golden Gate Protocol

BASIS

The protocol described below outlines the steps to be followed to perform ligation using the Golden Gate technique.

MATERIAL

REAGENTS

PROCEDURE

  1. Set up a restriction-ligation by pipetting into a tube 20 fmol (approximately 50 ng) of each level 0 module and of the vector, 2 uL 10x ligation buffer, 10U (1uL) of BsaI, and either 3U (1uL) of ligase for assembly of two to four modules or 20 U (1uL) HC ligase assembly of more than four modules (final volume of 20 ul).
  2. Incubate the restriction-ligation mix in a thermal cycler. For assembly of two to four level 0 modules, incubation for 60 to 120 minutes at 37ºC is sufficient. If more modules are ligated together, the incubation time is increased to 6h, or cycling is used as following: 20 min at 37ºC followed by 3 min at 16ºC, both repeated 50 times.
  3. Restriction-ligation is followed by a digestion step (5 min at 50ºC) and then by heat inactivation for 10 min at 80ºC. The final incubation step at 80ºC is very important and is needed to inactivate the ligase at the end of the restriction-ligation. Omitting this step would lead to relegation of some of the insert and plasmid backbone fragments when the reaction vessel is taken out of the thermal cycler, and would lead to a higher proportion of colonies containing incorrect constructs.
  4. Transform the entire ligation into chemically competent DH10B cells.

The following formula (from the NEB catalog) can be used: 1 ug of 1000 bp DNA fragment corresponds to 1.52pmol. Therefore, the volume of DNA to pipet (in ul) to have 20fmol is given by the equation: 20 (fmol) x size (bp) of the DNA fragment/concentration (ng/ul) x1520). Frequently, this formula will leads to volumes 1ul with standard miniprep DNA. In these cases, we recommend diluting the DNA to avoid pipetting errors.

Miniprep protocol

BASIS

Miniprep is a technique for the separation, extraction and purification of plasmid DNA. Generally, it is used to obtain cloned DNA, the storage of which is possible for later use. It is an essential step when testing cloning in bacteria, followed by enzymatic digestions and electrophoresis.

The protocol followed and described below is provided by GeneJET.

MATERIAL

REAGENTS

PROCEDURE

  1. Resuspend the pelleted cells in 250 pL of the Resuspension Solution. T to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. Note: ensure RNase A has been added to the Resuspension Solution.
  2. Add of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5. min to avoid denaturation of supercoiled plasmid DNA.
  3. Add 350 plL of the Neutralization Solution and mix immediately and thoroughiy by inverting the tube 4-6 times. Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial Iysate should become cloudy.
  4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
  5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Note. Close the bag with GeneJET Spin Columns tightly after each use!
  6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same. collection tube. Note. Do not add bleach to the flow-through
  7. Add 500 pL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
  8. Repeat the wash procedure (step 8) using 500 pL of the Wash Solution.
  9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
  10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 50 pL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
  11. Discard the column and store the purified plasmid DNA at -20*C.

Mix and Go Competent Cells protocol

BASIS

The bacterial ability to acquire DNA vectors (transformation) is natural in some cases and inducible in others. Although there are commercial companies that sell bacteria that are already competent, it is common in the laboratory to induce competence in the bacteria you have.

The following protocol is provided by Mix and Go.

MATERIAL

REAGENTS

PROCEDURE

The following procedure is for 50 ml E. coli culture in ZymoBroth or SOB medium. However, the volume can be adjusted according to your specific requirements.

  1. Use 0.5 ml of fresh, overnight E. coli culture grown in LB to inoculate 50 ml Zymobroth or SOB medium in a 500 ml culture flask. Shake culture vigorously (150-250 rpm) at the appropriate temperature until the OD (600 nm) is 0.4-0.6.
  2. Buffer preparation:
  3. Transfer the culture from Step 1 to ice. After 10 minutes, pellet the cells by centrifugation at 3000-3700 rpm for 10 minutes at 0-4ºC.
  4. Remove the supernatant and resuspend the cells gently in 5 ml ice-cold 1X Wash Buffer. Re-pellet the cells in Step 2.
  5. Completely remove the supernatant and gently resuspend the cells in 5 ml ice-cold 1X Competent Buffer.
  6. Aliquot (on ice) 0.1-0.2 ml of the cell suspension into sterile microcentrifuge tubes. Cells are now ready for transformation with DNA or can be stored below -70ºC for transformation at a later time.

Neb Turbo Competent (C2984H) transformation protocol

BASIS

Bacterial transformation is an essential step in genetic engineering. It allows the cell to adopt the plasmid to be cloned.

The protocol followed and described below is provided by NEW ENGLAND BioLabs.

MATERIAL

REAGENTS

PROCEDURE

  1. Thaw a tube of NEB Turbo CompetentE. coli cells on ice for 10 minutes.
  2. Add 1-5 ul containing 1 pg-100ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex
  3. Heat shock at exactly 42ºC for exactly 30 seconds. Do not mix.
  4. Place on ice for 5 minutes. Do not mix.
  5. Pipette 950 μl of room temperature SOC into mixture.
  6. Place at 37ºC for 60 minutes. Shake vigorously (250rpm) or rotate.
  7. Warm selection plates to 37ºC.
  8. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  9. Spread 50-100 ul of each dilution onto a selection plate and incubate 8-12 hours to overnight at 37ºC. Alternatively, incubate at 30ºC for 16 hours or 25ºC for 24 hours.

PCR protocol

BASIS

Polymerase Chain Reaction (PCR) is a technique in which, starting from a known DNA fragment, it is copied and amplified billions of times. The final product obtained is visualized on an agarose gel and provides qualitative evidence of the presence of that DNA fragment in the sample.

It is a sensitive and highly useful technique for obtaining and generating many copies of specific DNA sequences. To carry out this process, the use of an enzyme called Taq Polymerase is required, which is widely employed in these methods and their variations due to its ability to withstand high temperatures. This method provides recommendations to ensure a successful PCR.

MATERIAL

REAGENTS

PROCEDURE

Note: All materials must be sterile (autoclaved) and clean. It is preferible to preheat the termal cycler to 95oC.

  1. First, calculations are made to prepare the necessary reactions following Table 1. Always account for a few extra samples than the ones you have. This way, one will be for a negative control, another for the positive control, and the last one as a backup in case of evaporation or losses due to pipetting errors.
  2. In a tube, add the following components depending on the sample size.
    3. 10 μl 25 μl 50 μl 100 μl
    5x buffer 2 μl 5 μl 10 μl 20 μl
    dNTPs (2.5 M each) 0.8 μl 2 μl 4 μl 8 μl
    Forward primer 0.5 μl 1.25 μl 2.5 μl 5 μl
    Reverse primer 0.5 μl 1.25 μl 2.5 μl 5 μl
    Mg (2.5 mM) 1.2 μl 3 μl 6 μl 12 μl
    H2O 4.55 μl 11.375 μl 22.75 μl 45.5 μl
    Taq polymerase 0.05 μl 0.125 μl 0.25 μl 0.5 μl
    Template (10 – 25ng/μl) 0.4 μl 1 μl 2 μl 4 μl
  3. Add the reagents from Table 1 to a single PCR tuve and label it as the MasterMix

    NOTE: Place the Taq Polymerase at the end. Remove the enzyme from the freezer at the moment of use and immediately return it to the freezer.

    NOTE: Add the component with the highest volume first (i.e., water), and then continue adding them in descending order based on volume.

  4. Mix well with a pipette (up and down).
  5. In PCR tubes suitable for the thermal cycler, add 10 μl of the MasterMix (step 3).

    NOTE: The last two wells will be for the negative control (pure water) and the positive control..

  6. Spin down all these wells.
  7. Place the simples in the termal cycler and run the following program:
    Steps Temperature Time
    Desnaturation 95 ºC 10 minutes
    35 Cycles

    95ºC

    56ºC

    72ºC

    30 seconds

    30 seconds

    5 seconds
    Final extension 72ºC
    Cooling 4 - 10ºC infinite

    After completing the PCR, the product is analyzed on an agarose gel (electrophoresis).

Pomace treatment protocol

BASIS

This protocol is done almost in every sample preparation before any analytic technique. It’s used to transform the sample into a small and dry particle state. This way the compounds in the pomace are more accessible.

MATERIAL

PROCEDURE

  1. Put the pomace in the oven at 30ºC as scattered as possible. (It’s recommendable to use an oven with fan or some kind of air circulation to speed up the dehydration)
  2. Keep making weighs of the sample, until two consecutive weights are equal.
  3. Grind the pomace in the coffee grinder (180W).
  4. Sieve the pomace dust.
  5. The pomace dust that can't be completely sieved this could be grinded again.
  6. Repeat the last 3 steps until getting the desired sample quantity