Identification of Lactobacillus

Introduction:

For the genus identification of lactobacillus specific primers will be used.

Forward primer:XB5

Reverse primer:LBlMA1

Primers target the 16SrDna and 23SrDna region and amplify this region.

Amplicon size~250bp.

Basic steps:

1.Isolate pure culture of lactobacillus

2. Perform DNA extraction.

3. Use DNA in PCR using lactobacillus specific primers.

4. Required band size obtained.

5.Run the Gel.

PCR protocol

Reaction components are assembled as described below. The final volume should be 50 µL.

1.Thaw all reagents on ice

2.Assemble reaction mix into 50 µL volume in a thin walled 0.2 mL PCR tubes.

3.Add reagents in following order: water, buffer, dNTPs, Mg CL2, template primers, Taq polymerase.

4.Gently mix by tapping tube. Briefly centrifuge to settle tube contents.

Prepare negative control reaction without template DNA.

5.Prepare positive control reaction with template of known size and appropriate primers

Component Final Concentration/amount

water 50 µL
buffer 1 x TAE buffer
Taq polymerase 0.05 units/µL
dNTP mix 200 µM
MgCl2 0.1-0.5 mM
Forward primer 0.1-0.5 µM
Reverse primer 0.1-0.5 µM
Template 200 pg/µL
DMSO (optional) 1 to 10% w/v
PCR programing:

Initial denaturation:

It is done at 94°C for 10 minutes

Final denaturation:

It is done at 94°C for 1 minute

Annealing:

It is done at 55°C for 1 minute

Initial extension:

It is done at 72°C for 1 minute

Final extension:

It is done at 72°C for 10 minutes

Cooling:

The sample is cooled at 4°C.

GEL PREPARATION:

MATERIALS:

1.TAE buffer(1X)

2.Agarose gel(2 percent)

3.Ethidium bromide

4.Loading dye(if master mix is not coloured)

PROCEDURE:

Prepare 2% agarose gel by dissolving 1g of agarose powder in 50ml of 1X TAE buffer(as 2g is needed for
100ml so 1g for 50ml) in a conical flask.Dissolve agarose in a microwave until the solution becomes
clear.Let the solution cool to room temperature. When it comes to room temperature, add 3-5μl
ethidium bromide.Seal casting tray,insert comb into it and pour gel in it.Leave it to solidify for 15-20
minutes.

LOADING OF SAMPLE:

If master mix prepared previously is coloured,there is no need of loading dye.But if master mix is
colorless,then take 4μl amplicon+1μl loading dye and mix them.Now load it into wells.

In first well,load the ladder and then load sample in remaining wells.
Run the gel at 120 volts for 40 minutes.

After completion of time,take out the gel and observe under UV illuminator.Bands will bhi seen.

Record the result.

PRINCIPLE OF SETTING VOLTAGE:

Distance between cathode and anode x 5.

Result:

Ladder is observed in first first well. Samples were added

in the next well. Bands were observed.

Discussion:

Using PCR, a DNA sequence can be amplified millions or billions of times, producing enough DNA copies
to be analyzed using other techniques. For instance, the DNA may be visualized by gel electrophoresis,
sent for sequencing, or digested with restriction enzymes and cloned into a plasmid.

PCR is used in many research labs, and it also has practical applications in forensics, genetic testing, and
diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA
of patients (or from fetal DNA, in the case of prenatal testing). PCR can also be used to test for a
bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify
regions of its DNA from a blood or tissue sample.