Student PCR

GeNeiTM

Student PCR

GeNeiTM

GeNeiTM Student PCR Teaching Kit Manual

Cat No. KT44 KT44A KT44B

Bangalore Genei, 2007 Bangalore Genei, 2007

New Cat No. 106302 106303 106304

Revision No.: 00061204

Student PCR

GeNeiTM

Student PCR

GeNeiTM
CONTENTS
Page No.

Objective Principle Kit Description Materials Provided Procedure Observation & Interpretation

3 3 9 10 11 13

AGAROSE GEL ELECTROPHORESIS

Introduction Principle Procedure

17 17 20
22

ORDERING INFORMATION

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Objectives:
To perform PCR amplification of specific target sequence from genomic DNA. To analyze the amplified product by agarose gel electrophoresis.

Principle:
PCR Polymerase Chain Reaction is an in vitro method of enzymatic synthesis of specific DNA sequences, developed by Kary Mullis in 1983. It is a very simple and inexpensive technique for characterizing, analyzing and synthesizing any specific piece of DNA or RNA from virtually any living organism plant /animal /virus /bacteria. It exploits the natural function of the polymerases, present in all living things, to copy genetic material or perform Molecular Photocopying. PCR consists of three basic steps: Denaturation: During this step, the two strands melt open to form single stranded DNA and all enzymatic reactions stop. This is generally carried out at 92C 96C. Annealing: Annealing of primers to each original strand for new strand synthesis is carried out between 45C 55C. Extension at 72 C: The polymerase adds dNTPs complementary to the template at the 3 end of the primers. Since both strands are copied during PCR, there is an exponential increase in the number of copies of the gene.

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

These 3 steps are repeated 20-30 times in an automated thermocycler that can heat and cool the reaction mixture in tubes within a very short time. This results in exponential accumulation of specific DNA fragments, ends of which are defined by 5 ends of the primers. The doubling of number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula: Amplification = 2n, where n = No. of cycles PCR can amplify a desired DNA sequence of any origin, hundreds of millions of times in matter of hours, a task that would require days with recombinant technology. It is especially valuable because the reaction is highly specific, easily automated and very sensitive. It has thus revolutionized fields like: Clinical medicine Diagnosis of genetic disorders Forensic sciences Evolutionary biology

Factors affecting Amplification: I. Reaction components: Sample Volume: Most amplifications are performed at 20, 50, or 100 l volume in 0.2 or 0.5 ml microfuge tubes. Larger volumes do not allow adequate thermal equilibrium of the reaction mixture. Template DNA: Generally nanogram amount of plasmid DNA, or microgram amount of genomic DNA is used for PCR. Higher amounts of template DNA inhibits or results in non-specific amplification. Primers: PCR reaction needs two primers, a forward and a reverse primer. Primers are synthetic oligonucleotides usually ranging from 15 to 30 bases. Primers are designed such that at the 3 ends they do not have more than two bases complimentary to each other, as this results in PRIMER-DIMER formation. G+C content is in the range of 40-60%. The melting temperature (Tm) of both forward and reverse primers is usually the same. Low concentrations of primers result in poor yield of the specific product and high concentrations of primers may result in non-specific amplification. Optimal concentration of primers is between 0.1-1 M.

Bangalore Genei, 2007

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Deoxynucleotide-tri-phosphates: The final concentration of each dNTP (dATP, dGTP, dCTP & dTTP) in a standard amplification reaction is 200 M. It is important to keep the dNTP concentrations above the estimated Km of each dNTP (10 to 15 M) for best base incorporation. Taq DNA polymerase buffer: The 10X assay buffer contains 100 mM Tris- Cl (pH 9.0), 500 mM Potassium chloride, 15 mM MgCl2 and 0.1% w/v gelatin. Mg2+ is an essential cofactor required for the activity of Taq DNA Polymerase. Low concentration of Magnesium will result in no amplification and high amounts may lead to the production of unwanted products. Taq DNA Polymerase: Taq DNA polymerase is a 94 kD (Kilodaltons) thermostable DNA polymerase. Optimal temperature for Taq DNA Polymerase activity is 72C. It lacks 3 to 5 exonuclease activity but has 5 to 3 exonuclease activity and 5 to 3 polymerase activity. For most amplification reactions 1.5 to 2 units of enzyme is recommended as higher enzyme concentration leads to non-specific amplification.

II Temperature cycling: Temperature cycling parameters depend greatly on the template, primers and amplification apparatus. Initial denaturation step: Complete denaturation of template DNA at the start of the PCR reaction is of key importance. Incomplete denaturation results in inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. It is generally performed at 95C for 1-3 minutes, depending on the GC content of the template. If longer initial denaturation or temperature is necessary, then Taq DNA Polymerase can be added after this step as higher temperature may affect the enzyme stability. Initial denaturation step is performed only once at the beginning of the reaction. Denaturation: Subsequent denaturation steps are performed for a shorter time of 30 seconds - 1 minute at 94C for 30 cycles. This is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and completely denatures under these conditions. Certain additives like DMSO/glycerol/ formamide are used to facilitate DNA denaturation depending on the GC content. Annealing: The optimal annealing temperature is generally 5C lower than the melting temperature of primer-template DNA duplex, performed for 30 sec.-1minute. If non-specific products are obtained in addition to the expected product, the annealing temperature is optimised by increasing it stepwise by 1-2C.

Bangalore Genei, 2007

Bangalore Genei, 2007

Student PCR

GeNeiTM

Student PCR

GeNeiTM

Extension: Primer extension, resulting in synthesis of new DNA strand is carried out at 72C, which is optimal temperature for Taq DNA Polymerase activity. The amplification time is determined by the length of the sequence to be amplified. For every 1 kb target DNA, one minute time is recommended. Number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the product. Final extension: As DNA synthesis proceeds, it becomes less efficient as most of the components get used up. Hence, following the last cycle, enzyme is allowed to finish any incomplete synthesis by carrying out a final extension at 72C for 5 15 minutes.

Kit Description:
In this kit, reagents are provided to perform PCR using DNA purified from Serratia marcescens as the template. Two primers, i.e., forward primer and reverse primer are used to amplify a region of the template that results in a product of size 800 bp. The amplified product is then analyzed by running the sample on 1.5 % agarose gel along with a marker. The DNA marker is a 100 bp ladder wherein, 10 bands are seen ranging from 100 bp to 1 kb. KT44 : The kit is designed to carry out 10 PCR amplifications. The kit also includes electrophoresis equipment (ETS-1) required for Agarose gel electrophoresis. The kit is designed to carry out 10 PCR amplifications. The kit is designed to carry out 20 PCR amplifications. Electrophoresis equipment is required for KT44A and KT44B.

KT44A : KT44B : Note :

Duration of experiment: Approximately 4 hours.

Bangalore Genei, 2007

Bangalore Genei, 2007

Student PCR

GeNeiTM

Student PCR

GeNeiTM

Materials Provided:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.
Quantity Materials
10X Assay Buffer 10 mM dNTP Mix 100 bp ladder (ready to use) Forward Primer Reverse Primer Nuclease Free Water Template DNA Taq DNA Polymerase 2.5X Gel Loading Buffer Agarose 50X TAE Mineral oil PCR tubes

Note:
Read the entire procedure before starting the experiment. Agarose supplied is sufficient to prepare five 1.5% gels in case of KT44 & KT44A and ten 1.5% gels in case of KT44B. Hence, perform minimum of two PCR amplifications at a time. Agarose gel to be prepared by addition of Ethidium bromide at a concentration of 0.5 g/ml from a stock of 10 mg/ml (refer Agarose Gel Electrophoresis). 100 bp ladder supplied is ready to use and can be loaded directly onto agarose gel.

KT44/44A
(10 reactions)

KT44B
(20 reactions)

Store
-20C -20C -20C -20C -20C -20C -20C -20C -20C RT RT RT RT

50 l 30 l 50 l 10 l 10 l 1 ml 10 l 10 l 0.25 ml 5g 20 ml 0.5 ml 10 Nos.

0.1 ml 60 l 0.1 ml 20 l 20 l 1 ml 20 l 20 l 0.25 ml 10 g 40 ml 1 ml 20 Nos.

Procedure:
Setting up PCR 1. Add the following reagents to the PCR tube in the following order: Sterile Water 10X Assay Buffer 10 mM dNTP Mix Template DNA (100 ng/l) Forward Primer (100 ng/l) Reverse Primer (100 ng/l) Taq DNA Polymerase (3 U/l) Total reaction volume 38 l 5 l 3 l 1 l 1 l 1 l 1 l 50 l

Materials Required:
Equipment : Thermocycler, UV-transilluminator. Reagents : Distilled Water, Ethidium Bromide. Other Requirements : Crushed Ice/Genei cooler, Tips, Gloves, Micropipette.
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2. Mix the contents gently and layer the reaction mix with 50 l of mineral oil, to prevent evaporation. Note : Mineral oil need not be added if the Thermocycler is equipped with a heated lid.
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Student PCR

GeNeiTM

Student PCR

GeNeiTM

PCR Amplification 3. Carry out the amplification in a thermocycler for 30 cycles using the following reaction conditions:
Initial Final Denaturation Denaturation Annealing Extension Extension 94C 94C 48C 72C 72C 1 minute 30 sec. 30 sec. 1 min. 2 min.

Observation:
Compare the amplified band with 100 bp ladder and note down the size of the fragment. Also observe for presence of any other bands other than the amplified product
1 2

For 30 cycles

Lane 1 : Lane 2 :

Analysis on Agarose Gel 4. Following PCR amplification, add 5 l of Gel loading buffer to each of the PCR tubes. 5. Tap the mixture thoroughly and wait for a few seconds for the 2 layers to separate. 6. Carefully pipette out 15 l of reaction mixture (avoiding mineral oil layer) and load onto 1.5% agarose gel. 7. Load 10 l of the ready to use marker provided. Note down the order in which the samples have been loaded. 8. Run the samples at 100 volts for 1-2 hours till the tracking dye (bromophenol blue) reaches 3/4th of the length of the gel. 9. Visualize the gel under UV transilluminator.

100 bp DNA Ladder (100 bp to 1000 bp). PCR Amplified 0.8 kb fragment electrophoresing alongside 0.8 kb fragment of 100 bp ladder

Fig 1: PCR amplified product run on 1.5% Agarose gel (Stained with EtBr)

Interpretation:
As observed on agarose gel, PCR amplification of the template using specific primers results in a specific product of a particular length. The conditions have been optimised to give a highly specific product of 800 bp as is observed by the absence of any non-specific products. Altering these conditions would result in non-specific amplification. PCR was carried out with 100 nanograms of template, having approximately 1000 copies of the target sequence. Following PCR, the product yield is in microgram quantity, which is approximately a million copies of the target sequence, highlighting the fact that PCR is a very sensitive technique.

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Agarose Gel Electrophoresis

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Introduction:
Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their molecular weight and is an intrinsic part of almost all routine experiments carried out in molecular biology. The technique consists of 3 basic steps: Preparation of agarose gel Electrophoresis of the DNA fragments Visualization of DNA fragments

Principle:
Preparation of Agarose Gel: Agarose is a linear polymer extracted from seaweeds. Its basic structure is shown in the figure:

HO

CH2O
OH

HO
Fig: Basic unit structure of Agarose. Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution is then poured into a mould and allowed to solidify. As it cools, agarose undergoes polymerisation i.e., sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which is determined by concentration of agarose.

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Electrophoresis of DNA fragments: Electrophoresis is a technique used to separate charged molecules. DNA is negatively charged at neutral pH and when electric field is applied across the gel, DNA migrates towards the anode. Migration of DNA through the gel is dependent upon: 1. Molecular size of DNA 2. Agarose concentration 3. Conformation of DNA 4. Applied current Matrix of agarose gel acts as a molecular sieve through which DNA fragments move on application of electric current. Higher concentration of agarose gives firmer gels, i.e., spaces between cross-linked molecules is less and hence smaller DNA fragments easily crawl through these spaces. As the length of the DNA increases, it becomes harder for the DNA to pass through the spaces, while lower concentration of agarose helps in movements of larger DNA fragments as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel. Two commonly used dyes are Xylene cyanol and Bromophenol blue that migrate at the same speed as double stranded DNA of size 5000 bp and 300 bp respectively. These tracking dyes are negatively charged, low molecular weight compounds that are loaded along with each sample at the start of run, when the tracking dye reaches towards the anode, run is terminated.

Visualization of DNA fragments: Since DNA is not naturally coloured, it will not be visible on the gel. Hence the gel, after electrophoresis, is stained with a dye specific to the DNA. Discrete bands are observed when there is enough DNA material present to bind the dye to make it visible, otherwise the band is not detected. The gel is observed against a light background wherein DNA appears as dark coloured bands. Alternatively, an intercalating dye like Ethidium bromide is added to agarose gel and location of bands determined by examining the gel under UV light, wherein DNA fluoresces. Note: Ethidium bromide must be handled carefully as it is a mutagen and a carcinogen. Wear gloves while handling EtBr solution & gels stained with EtBr.

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Procedure:
Preparation of 1.5% Agarose Gel 1. Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer. (For one experiment, approximately 200 ml of 1X TAE is required. Make up 4 ml of 50X TAE to 200 ml with distilled water). 2. Weigh 0.75 g of agarose and add to 50 ml of 1X TAE. This gives 1.5% agarose gel. 3. Boil till agarose dissolves completely and a clear solution results. 4. Meanwhile place the combs of electrophoresis set such that it is approximately 2 cm away from the cathode. 5. Pour the agarose solution in the central part of tank when the temperature reaches approximately 60C. Do not generate air bubbles. The thickness of the gel should be around 0.5 to 0.9 cm. Keep the gel undisturbed at room temperature for the agarose to solidify. 6. Pour 1X TAE buffer into the gel tank till the buffer level stands at 0.5 to 0.8 cm above the gel surface. 7. Gently lift the combs, ensuring that wells remain intact.

Electrophoresis 8. Connect the power cord to the electrophoretic power supply according to the convention red: anode, black: cathode. 9. Load the samples in the wells in the desired order. 10. Set the voltage to 50 V and switch on the power supply. 11. Switch off the power when the tracking dye (bromophenol blue) from the well reaches th of the gel. This takes approximately one hour. Staining Procedure to Visualize DNA 12. Prepare 1X staining dye by diluting 6X dye (1:6) with distilled water. (Approximately 50 ml of 1X staining dye is required for one experiment. Therefore, make up 8 ml of 6X dye to 48 ml with distilled water). 13. Carefully transfer the gel (from gel tank) into a tray containing 1X staining solution. Make sure that the gel is completely immersed. 14. For uniform staining, place the tray on a rocker for approximately one hour or shake intermittently every 10 to 15 minutes. 15. Pour out the staining dye into a container. (The dye can be reused twice). Destain the gel by washing with tap water several times till the DNA is visible as a dark band against a light blue background. Note: Alternatively, Ethidium bromide can be used for visualizing DNA fragments. Add Ethidium bromide to molten agarose to a final concentration of 0.5 g/ml (from a stock of 10 mg/ml in water), when temperature is around 50C. Mix and cast the gel. After electrophoresis, DNA samples can be visualized under UV light, they appear fluorescent. No destaining is required in this case.

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Student PCR

GeNeiTM

Student PCR

GeNeiTM

Ordering Information
Product Size Cat #

GeNeiTM Student PCR 1 Pack KT44 Teaching Kit (Consumables for 10 experiments & Elpho Kit (ETS 1)) GeNeiTM Student PCR Teaching Kit (Consumables 10 experiments) GeNeiTM Student PCR Teaching Kit (Consumables 20 experiments) 1 Pack KT44A

1 Pack

KT44B

Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com

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