Laboratory Manual

 

 

 

 

Laboratory Practices and Safety

 

Genomic DNA isolation

 

Gel Electrophoresis

 

DNA Quality Check

 

Polymerase Chain Reaction for SSRs

 

SSR gel visualization

 

PCR for Random Amplified Polymorphic DNAs

 

 

 

 

Laboratory Practices & Safety::

 

 

All the enzyme used in these procedures are very expensive! Always keep the enzyme on ice and remove the enzyme from the freezer only before starting the required step of your experiment!

 

Laboratory equipment is also very expensive and, sometimes (e.g. in the case of centrifuges) potentially dangerous.

 

Please don't use any equipment you are unfamiliar with without asking for help!

 

Ethidium bromide is a carcinogen. Gloves should be worn at all times.

 

Ultraviolet light is dangerous to the eyes. Goggles should be worn unless other shielding is available.

 

Of course, hands should be washed after any laboratory practices and especially before any food is eaten.

 

 

 

 

Genomic DNA isolation

(Method of Gawel and Jarret 1991)

 

Working with gloves on at all times,

 

Pre-chill plant leaves (0.15 g) at -80C and crush in liquid nitrogen with a mortar and pestle.

Pour (scrape) homogenate into a microfuge tube.

 

Add a pre-heated (65oC) extraction buffer (0.5 ml) containing 100 mM Tris-HCl (pH 8); 1.4 mM NaCl; 20 mM EDTA; 0.1% mercaptoethanol and 2% CTAB to crushed leaf material.

 

(Alternatively homogenize 0.15 g of leaf tissue in a labeled microfuge tube with 0.5 ml of extraction buffer.)

 

Incubate the mixture at 65C for 30 min followed by addition of 0.4 ml of chloroform:isoamyl alcohol (24:1) mixture and incubate the homogenate for 15 min at room temprature.

Centrifuge for 5 min at maximum speed (14,000 rpm) in a microfuge at room temperature.

 

Carefully remove 0.5 ml of the upper aqueous phase into a new tube (avoid transferring any material form the interface) with a 1 ml micropipette and add an equal volume of ice-cold isopropanol to precipitate the DNA. Stand on ice for 10 minutes.

 

Centrifuge at maximum speed for 5 min at room temperature to collect the DNA.

 

Wash the DNA pellet by adding 0.5 ml 70% ethanol, and centrifuging at maximum speed for 2 min.

 

Dry the pellet in a vacuum centrifuge (SpeedVac) and re-suspend in 100 µl sdH2O (sterile distilled water).

 

 

Remove any contaminating RNA by addition of 1 µl of a 10 µg/ml stock solution of RNase and incubate at 37C for 30 min.

 

Recover DNA by the addition of 1/10 volume of 3M sodium acetate (pH 6.8) and 2 volumes of 96% ethanol to the DNA containing solution.

 

Incubate on ice for 10 min

 

Centrifuge at maximum speed for 5 min at room temperature, to pellet the DNA.

 

Wash and dry DNA as above and finally dissolve in 50 µl sdH2O.

 

 

 

 

 

Gel Electrophoresis

 

1)             A 1 % agarose solution in 1 x TAE buffer will be used (10X TAE is 108 g/l Tris Base, 55 g/l acetic acid, 9.3 g/l EDTA; pH should be 8.0 or 8.1). This is the same buffer that will be in the electrophoresis tank. If the gel is made with a different buffer then it will run aberrantly and the molecular weight calculations and conclusions will not be valid.

2)             Add 100ml 1 x TAE buffer to a 250 ml Erlenmeyer flask.

3)             Calculate the amount of agarose needed to make a 1 % gel.

x g agarose/ 100 ml = 1 %

4)             Weigh out this quantity of agarose and add it to the flask. Leave the flask to stand for 5 minutes to hydrate. This is important to avoid the solution boiling over when heated in the microwave.

5)             Place a foam bung on top of the flask and place in a microwave. Heat on medium heat until all the agarose has gone into solution. It is best to stop the microwave at minute intervals and swirl the flask to ensure thorough mixing of the agarose. Be careful when the agarose is nearly melted that it does not suddenly boil when you mix it. Severe burns can occur without due care at this stage. You should always wear a glove when swirling heated flasks.

6)             Allow the agarose to cool to below 55C either on the bench or in a waterbath. If the agarose is too hot the gel trays will deform.

7)             Add 1 ml of a 10 mg/ml solution of ethidium bromide to the gel at this point and swirl to mix.

8)             Place the gel tray in the casting tray and insert the combs

9)             Pour the gel to a thickness of 4-5 mm. It is important that the agarose is not too hot, otherwise it will tend to warp the casting tray.

Wear gloves when handling the gel at all subsequent stages. Care should be taken when dealing with ethidium bromide-- wear gloves. Ethidium bromide is a mutagen and a suspected carcinogen.

10)          Allow the gel to set. It will become translucent all over when completely set. You will be able to notice the change from transparency to opaqueness at the edges first, but wait until the whole gel has set before removing the comb.

11)          Remove the gel tray from the casting tray and place in the gel box. Take care that the gel does not slip off the gel tray.

12)          Place the tray on the center support and add more 1 x TAE buffer until the gel is just covered.

13)          Carefully remove the comb from the gel. Convention dictates that the "top" of a gel is where the wells are. The gel is now ready! Make sure that you have placed the gel in the tray in the correct orientation Ð DNA is negatively charged and runs towards the positive electrode (Red).

 

DNA Quality and quantity check

 

To test for the quality and amount of isolated DNA, a sample of isolated DNA (5 µl) is run on the 1% agarose gel in TAE buffer (0.04 M Tris-acetate; 1 mM EDTA, pH 8) that was prepared as described above.

 

Place 5 µl of your DNA in a microfuge tube, add 5 µl of water and 2 µl of loading dye and place all the solution into one of the wells in the gel. In one lane place a DNA concentration standard (usually a known amount of lambda DNA). A range of lambda DNA amounts (50ng, 100ng, 250ng and 500ng) can be used to get a more accurate estimation of the DNA concentration. We will use these 4 concentration standards

 

Run the gel at 100V for 90 minutes

Determine the DNA quality on a UV-transilluminator, photograph with a Grab-IT system (Vacutec, USA) and determine the DNA concentration of samples visually using the λ DNA amounts for comparison.

 

Dilute an aliquot of your DNAs to a concentration of 10ng/µl for the PCR reactions.

 

 

 

 

Gawel N.J. and Jarret R.L. 1991. A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Molecular Biology Reporter 9: 262-266.

Polymerase chain reaction (PCR)

 

The polymerase chain reaction is a three-step process, which is repeated in several cycles. The three steps are denaturation, annealing and extension. In the first of the chain reaction, denaturation, the two DNA strands are separated by heating the DNA to 94oC. Heat treatment breaks the relatively weak bonds between the DNA bases yielding two long single strands. In the second step, annealing, two primers attach themselves to the single strands. These primers are small, synthetic stretches of single-stranded DNA each about 20 base pairs long. They are selected so that they are complementary to the two single strands. The primers track down and bind themselves to the region being sought by framing the target sequence within the long DNA strand. The annealing temperature is usually in the range of 50-65oC depending on the length and composition of the primer. Once the primers have attached themselves, two short stretches of double-stranded DNA are generated. In the third step, extension, these short stretches serve as starting blocks for the enzyme Taq polymerase. Starting of the 3'-end of the primers, the enzyme adds the nucleotides complementary to the template at about 72oC, linking them together. It extends the primers in the direction of the target sequence, thereby making a double strand out of the two single strands. From the original double strand, there are now, at the end of this first cycle, two new DNA double strands identical with the first one. The three-step cycle can now be repeated as often as necessary where only the sequence flanked by the two primers is amplified. The new DNA sequences provide the template in the next cycle for the creation of new strands, resulting in four in the next cycle and then in 8, 16, 32 copies and so on.

 

 

All the enzyme used in this experiment are very expensive! Always keep the enzyme on ice and remove the enzyme from the freezer only before starting the required step of your experiment!

 

To carry out the PCR reaction, use small PCR tubes with your genomic DNA as template and the primer(s) that will amplify your DNA sequence. The PCR reaction is carried out in 50 µl volumes, which contains your genomic DNA, 15 ng of primer, 100 mM of each dNTP, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl2 and 2 units of Taq polymerase.

 

Each of you will use the maize DNA sample that you extracted and one of the land races with two microsatellite primer sets (that is you will set up 4 PCR tubes).

 

Recipe:

 

Stock

Final conc

µl Single Reaction

µl Cocktail

Genomic DNA

 

 

 

Primer 1

 

 

 

Primer 2

 

 

 

dNTP

 

 

 

Tris-HCl

 

 

 

MgCl2

 

 

 

Taq

 

 

 

 

Place all tubes into the thermal cycler and use the following DNA amplification programme:

 

(i)             94C for 5 minutes x 1 cycle;

(ii)           94C for 1 minute;

(iii)          55C for 1 minute, 72C for 1 minute x 40 cycles;

(iv)          72C for 7 minutes x 1 cycle;

(v)           hold at 4C until ready to load onto gel.

 

The amplification will last about 4 hours!

 

 

SSR product visualization

 

For the SSR technique pour a 3% super-fine agarose gel (50 or 100 ml using 1xTAE buffer, ask your supervisor for the exact ml to be used which depends on the gel size). The demonstrator will show how to melt the agar and to pour the gel. Before pouring the gel add 2 µl ethidium bromide and mix gently the agar with the ethidium bromide (10mg/ml). Why do you add ethidium bromide to the mixture?

 

Add 5 µl of the loading dye to your PCR reaction. Load 20 µl of the mixture into one of the wells in the gel. All the reactions with the same set of primers should be run in adjacent lanes to make size comparisons easier. Run the gel at 120mV for 90 minutes. DonÕt let the dye run out of the gel. Place gel on UV screen by wearing gloves and write down your observation. Also take a photo from your gel for your protocol; the demonstrator will help you in making a photo.

 

 

 

Random Amplified Polymorphic DNAs (RAPDs)

 

RAPDs are different from all other PCR reactions in that only a single short primer is used. The reaction depends on there being the same short sequence present in inverse orientation within a short enough stretch of DNA that can be amplified by PCR. The optimum length for these primers is ten nucleotides. These primer are used with no other knowledge of the sequence of the DNA being tested. Therefore with some of the randomly chosen decanucleotides no sequences are amplified. With others, the same length products are generated from DNAs of different individuals. With still others, patterns of bands are not the same for every individual in a population. It is these variable bands that are called random amplified polymorphic DNA (RAPD) bands.

<2kb in length

The reactions are set up in a similar fashion to those for the SSRs, contaning a DNA template, dNTPs, the correct PCR buffer but only a single primer.

The primers to be used are:

OPA11

OPA15

OPB15

OPC8

OPC10

140

C11

The essential steps are:

  1. 1.     A mastermix containing the dNTPs, PCR buffer and Taq polymerase will be prepared.
    1. 2.     Each individual will prepare 4 PCR tubes, one from the tomato DNA that they prepared and three from the control tomato DNAs
      1. 3.     Label each of the tubes
        1. 4.     To each of these tubes will be added :
            1. a.     1μL DNA solution
              1. b.     1μL of the appropriate primer
                1. c.     48μL of the mastermix
                1. 5.     Place all the tubes in the thermocycler
                  1. 6.     The reaction will proceed with the following parameters:

                    2. (i)             95C for 4 minutes x 1 cycle;

                    (ii)           35C for 30 sec, 72C for 90 sec, 95C for 40 cycles

                    (iii)          72C for 7 minutes x 1 cycle

                    1. 7.     Add 10μL of loading dye to each of your tubes
                      1. 8.     Prepare and pour a 1.5% agarose gel in TAE buffer with 3μL of ethidium bromide solution added per 100mL of gel solution
                        1. 9.     After the gel has set place in gel tank, make sure that the gel is completely submerged in buffer and add 5μL of the 100 base pair ladder molecular weight marker to each row of wells
                          1. 10.  The samples will be loaded on the gels so that all the samples that were amplified with a given primer are run in adjacent lanes.
                            1. 11.  Load 25μL of the sample plus loading dye into each well
                              1. 12.  Run the gel at 120 volts for 90 minutes
                                1. 13.  Photograph gel
                                  1. 14.  Analyse results