Team:MRIIRS FARIDABAD/Experimentation Protocols

Contribution

Protocols

Agarose Gel electrophoresis


It is a way out for separating different DNA fragment lengths out of a mixture. The path length or the distance travelled by the gel is based on the charge is to mass ratio of that particular DNA. Movement of negatively charged DNA fragments is from anode (negative terminal) to cathode (positive terminal). Also, Agarose forms the porous matrix through which DNA fragments pass and resolve as per their size or length. EtBr is added along with Ag arose that is used for visualization of DNA in the gel under UV.

Material Required:


  1. Glassware

  2. Horizontal gel electrophoresis unit

  3. TAE buffer 50X stock (1 liter)

Components Mass/volume
Tris base (2M) 242 g
EDTA (0.5 M and pH=8.0) 100 ml
Acetic Acid 57.1 ml
MilliQ Upto 1000 ml/1 liter
  • EtBr: Ethidium Bromide

  • Power Pack

  • UV trans illuminator

  • Gel Doc for obtaining the image.

  • Gel loading Dye.

    • Component Concentration
    Glycerol 30% (v/v)
    Bromophenol Blue 0.25% (w/v)
    Xylene Cyanol FF 0.25% (w/v)

  • 100 bp ladder, 25 bp ladder (selection of ladder depends on the size of gene amplified)


    • PROCEDURE

    1. The amount agarose to be used for casting a gel is based on the resolution that is required for resolving the desired DNA fragment length.

      The correlation is as follows:

      Size Range(bp) Final Agarose Concentration(%age)
      1000-30000 0.5
      800-12000 0.7
      500-10000 1
      400-7000 1.2
      200-3000 1.5
      50-2000 2

    2. For 8 well casting tray, 50 ml of agarose is sufficient to be solidified. We prepared the solution in 1X TAE buffer and heated it in Microwave Oven to solublize the agarose in Buffer and solidified along with comb inserted in the tray.

      *** Make sure there are no bubbles in the tray before solidification. ***

    3. After solidification, the tray is transferred to the buffer within the electrophoresis unit and comb is removed gently.

    4. After removing the comb, samples must be loaded and power supply is switched in with 80 volts as initial voltage.

    5. After 50-60 minutes observe the gel loading dye, if it has crossed ¾ of the length of gel, then switch of the gel.

    6. Extract out the casting tray from the unit and observed under UV and take the image in Gel Doc.

      For taking image, Image Lab software from BIO-RAD was used.

    Restriction Digestion and Ligation

      Restriction digestion of insert (ADA-AlkB Promoter and Reporter) and vector.

      1. For insert

        2. Component Volume ( μl )
        Insert Volume as per 1000 ng
        Enzyme 1 (Hind III) 1
        Enzyme 2 (Eco RI) 1
        Buffer suitable to both enzymes 5
        Add NFW to 50

        Component Volume ( μl )
        Insert Volume as per 1000 ng
        Enzyme 1 (BamHI) 1
        Enzyme 2 (Eco RI) 1
        Buffer suitable to both enzymes 5
        Add NFW to 50

        Component Volume ( μl )
        Insert Volume as per 1000 ng
        Enzyme 1 (BamHI) 1
        Enzyme 2 (HindIII) 1
        Buffer suitable to both enzymes 5
        Add NFW to 50

        After preparing the mix, spin the tubes and incubate them on 37° C at least for 1 hour.

        • Whole process to be done at ice.

        • Use one tip for a particular enzyme. Never use same tip in two different enzymes

        • Don’t insert the tip into the enzyme. Aspirate the same from surface.


      2. For vector

        3. Component Volume ( μl )
        Vector Volume as per 1000 ng
        Enzyme 1 (HindIII) 1
        Enzyme 2 (BamHI) 1
        Buffer suitable to both enzymes 5
        Add NFW to 50

        After preparing the mix, spin the tubes and incubate them on 37° C at least for 1 hour.

        • Whole process to be done at ice.

        • Use one tip for a particular enzyme. Never use same tip in two different enzymes

        • Don’t insert the tip into the enzyme. Aspirate the same from surface.


      Run the digested products on the agarose gel and do the gel elution

      For Gel elution, MDI Gel Extraction Kit was used. Protocol, as given with the kit, was followed.

      Concentration of pCMV-6-AC-His (digested) after elution: 18 ng/µL

      Concentration of end to end ligated insert (digested) after elution: 9 ng/µL

      Ligation

      The ligation reaction setup required some calculations to be done which are mentioned below in the form of screenshot. The software used for these calculations was LIGATION CALCULATOR- insilico from Universität Düsseldorf.

      First, end to end ligation is conducted to make it a complete insert. This will require 1:1 molar ratio of ADA-AlkB promoter and Reporter.

      ADA-AlkB Promoter (size in kb)/Reporter (size in kb) } X Molar ratio = ng of ADA AlkB promoter/ng of Reporter

      Promoter size was 120 bp and reporter size with terminator was 720 bp. This showed that 50 ng of reporter was used against 8.3 ng of ADA AlkB promoter. This gave the resulting concentration of the ligated product after digestion to be 9 ng/µL.

      Result of the above calculation:

      1. For calculating the volume of insert to be used for ligation, we need to divide the required amount by the concentration per µL of the eluted insert. As per this, the volume to be used will be 12.57/9 µL i.e. 1.4 µL.

      2. For calculating the volume of insert to be used for ligation, we need to divide the required amount by the concentration per µL of the eluted insert. As per this, the volume to be used will be 12.57/9 µL i.e. 1.4 µL. Similarly the volume of vector to be used can also be calculated: As per this, the volume to be used will be 50/18 µL i.e. 2.7 µL. The mix for ligation will be as follows:

      Component Volume ( μl )
      Vector 2.7
      Insert 1.4
      T4 DNA ligase 1
      Ligation buffer (10X) 1.5
      Add NFW to 15

      Leave the mix at room temperature for 1 hour or at 16°C overnight to get the ligation done.


      Preparation of competent cells

      Solutions and Reagents:

      1. 0.1 M MgCl2
      2. 0.1 M CaCl2
      3. Glycerol
      4. Nutrient Broth (Luria berttini broth)
      5. Centrifuge
      6. Incubator with shaker
      7. Ice in ice bucket
      8. Glassware
      9. Laminar air flow
      Procedure:

      1. Inoculate DH5-α E. coli in 25 ml broth and keep it on shaker incubator at 37° C overnight

      2. Growth will be observed in the culture. Take 100µL of that culture and inoculate 25ml LB broth as a subculture

      3. Retrieve the culture inoculated when OD reaches 0.4-0.6.

        **This usually takes around 3-4 hours. ***

      4. Centrifuge the obtained culture at 4000 rpm for 10 minutes at 4° C.

      5. Decant the supernatant and add 24 ml of MgCl2 (0.1 M) and 6 ml of CaCl2 (0.1 M) to the pellet obtained. It should be noted that both of the solutions should be ice-cold.

        ** The process of adding solutions should be done on ice.***

      6. Resuspend the pellet using pipette and incubate for 15 minutes on ice.

      7. After incubation, recentrifuge at 4000 rpm for 10 minutes at 4° C.

      8. Decant the supernatant, and then add 1.6 ml CaCl2 and 400µL of Glycerol. (all to be done at ice).

      9. Resuspend the same with pipette.

      10. Make 50 µL aliquots in centrifuge tubes and store at -80° C

        *** The centrifuge tubes should also be kept at 4° C before use. ***


      Transformation
      Material Required:

      • Shaking incubator at 37 °C

      • Stationary incubator at 37 °C

      • Water bath at 42 °C

      • Ice bucket filled with ice

      • Microcentrifuge tubes

      • Sterile spreading device

      • LB agar plate (with appropriate antibiotic)

      • LB media

      • competent cells

      • DNA to transform


      Procedure:

      1. Thaw the competent cells taken out of -80℃ at ice for 20-30 minutes

      2. After thawing the competent cells add 1-5µL of desired DNA (usually 10pg-100ng) into 50µL vial of competent cells. Gently mix by flicking the vial’s bottom.

      3. Place the competent cell/DNA mix on ice for 20 minutes.

      4. Give a heat shock to cells by placing the vials into a 42℃ water bath for 60-90 s by immersing around ½ to 2/3rd of the vial in water bath.

      5. The heated vials should be placed back on to ice for 2-3 minutes.

      6. Add 250-1000µL LB or SOC media (without antibiotic) to the bacteria and grow in 37℃ shaking incubator for 45 minutes.

        ***45 minutes incubation allows the bacteria to produce the antibiotic resistance proteins that are encoded in the plasmid backbone which will help these to grow on antibiotic resistant plates. Ampicillin resistance is an exception to it.

      7. After incubation, the vial should be centrifuged at 2000 rpm for 10 minutes and aspirate the supernatant such that only 50 µL of supernatant remains in the vial.

      8. Resuspend the pellet in that 50µL and spread it on desired antibiotic resistant plate using a sterile spreader.

        ** This is done to lower down the culture volume without decreasing the number of cells. If agar plates don’t dry before cells begin dividing, the bacteria will diffuse and won’t grow as colonies.**

      9. Incubate plates at 37℃ overnight.