Biology

PLANT VIROLOGY LAB TRAINING REPORT

MANNE SHIVA

Amplification of TuMV-VPg gene

1. INTRODUCTION:

Turnip mosaic virus (TuMV) is a Potyvirus of the family Potyviridae that causes diseases in cruciferous plants, among others. The virus is usually spread by 40-50 species of aphids in a non-persistent manner. Infected plants, especially the natural hosts, show symptoms such as chlorotic local lesions, mosaic, mottling, puckering or rugosity. TuMV is a positive-sense single stranded RNA virus, consisting of a non-enveloped, helical capsid that is filamentous and flexuous, with an average length of 720 nm. In this lab training we isoloate the TuMV VPggene and introduce into a new vector and then check for the transformation in E.coli followed by yeast transformation and western blotting.

2. MATERIALS AND METHODS

2.1.1 PCR AMPLIFICATION:

Water, 5X Phusion buffer, 10mM dNTP;’s, template and phusion enzyme, EcoRI, Sal1

PCR is set to the following conditons and run for 30 cycles.

Initial: 98C, 30sec

Denaturation: 98C 10sec

Annealing: 66c, 10sec

Elongation: 72C, 10sec

Final: 72C, 10min

PCR reaction mixture

PCR MIXTURE:

H2O : 33.5ul
5X buffer : 10ul
10mM dNTP : 1ul
Primer F : 2ul
Primer R : 2ul
Template: 1ul
Phusion : 0.5ul

After the PCR is finished, the genome is run on agarose gel electrophoresis to check whether the gene is amplified or not. We used EZNA gel extraction kit for purification of PCR product.

2.1.2 Restriction Digestion of the vector and gene:

The first step in cloning a DNA insert into a plasmid vector is cutting both vector and insert DNA with the appropriate restriction enzyme(s) to generate compatible ends. This may be a simple single digestion or a double digestion with two enzymes in the case of directional cloning. A partial digestion may be needed in situations where there is a lack of suitable sites to use and the restriction enzyme cuts more than once in the molecule.

We used EcoRI and SalI or XholI restriction digestion enzymes in this experiment for the vectors pGADT7 AD (7987bp) and pGBKT7(7.3kb) respectively.

We used yeast two hybrid vectors in this training. Vectors used are

1) pGADT7 vector harboring GAL4 activation domain

2) pGBKT7 vector harboring GALA4 DNA binding domain.

The two vectors and the gene are digested using the restriction enzymes asa mentioned above.

The reaction mixture of the digested sample is

Plasmid 1(40ul) Plasmid 2(40ul) Insert DNA(40ul)
10x Smart buffer 4ul 4ul 4ul
DNA or vector 6ul 6ul 30ul
Water 29ul 29ul 5ul
Restriction enzyme XholI 1ul SalI 1ul SalI 1ul

We used heat block method for the restriction digestion. We incubated the digestion mixtures in heat block at 37C for 90min. After the incubation time, the digestion mixture is made to 70ul using 1ul of EcoRI, 3ul of buffer and 26ul of water. This mixture is again incubated for 90min on heat block at 37C.

After the last restriction digestion, the vectors needs to be treated with CIAP enzyme to remove any phosphate groups present in the mixture which enhances the ligation.

1ul of CIAP enzyme is added to all the samples and incubated at 37C for 30min.

VMR omega gel extraction kit was used to purify the samples.

2.1.3 Ligation:

Ligation mixture:

Vector DNA: 2µl

Insert DNA: 1µl

Ligase 10X buffer: 1 µl

Promega T4 DNA ligase: 0.3 µl

Water: 5.7 µl

As a negative control, insert is not added in to the both the reaction mixture.

The above mixture is incubated at 12C overnight.

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2.1.4 E.coli Transformation

Transformation of bacteria is a process by which foreign plasmid DNA is taken up by bacteria. For this to occur the bacterial cell membrane must be made leaky and this is done by treating the cells with a mixture of divalent cations rendering their membranes temporarily permeable. Bacteria cells which are treated this way and are then competent to take up DNA are called competent cells. When competent cell are placed in solution containing plasmid, plasmid molecules can pass through the cell membrane into the cells. The process is called transformation due to the change (transformation) of the genetic makeup.

We use heat shock method for transfer of ligation mixture in the Ecoli. Competent cells DH5α is used.

Kanamycin and ampicillin plates using readymade LB media. Ligation mixture is chilled and 5ul of it is added to 50ul of competent cells. Gently mix by pipetting up and down to mix the cells and DNA. Do not vortex. Then the mixture is placed on ice for 30min followed by heat shock at 42C for 90 seconds. The mixture is transferred to ice and 600ul of LB is added to the tube. Tubes are rotated at 250rpm for 60 min at 37C and then spread across the selection plates using 50-100ul of the ligation mixture.

Plates are incubated at 37C overnight.

2.1.5 Colony PCR.

After the incubation of the selection plates, we saw growth in the plates and we choose some colonies to check whether the insert is present in Ecoli or not. We used EcoRI and SALI to check the presence of the insert DNA.

The reaction mixture of colony pcr :

1X 10X
Water 19.75ul 197.5ul
Dynazyme buffer 2.5ul 25ul
10mM dNTPS 0.5ul 5ul
Primer A 1ul EcoRI 10ul
Primer B 1ul SalI 10ul
Template DNA 0ul no template is used 0ul
Dynazyme II DNA polymeras 0.25ul 2.5ul

PCR reaction is set to:

Initiation: 94C for 5min

Denaturation: 94C for 30 sec

Annealing: 52C for 30sec

Elongation: 70C for 45 min

Termination: 70C for 5 min

The amplified samples are then run in 1% agarose gel at 140V for 20min.

2.1.6: Plasmid isolation

Cultures are grown overnight in ampicillin and kanamycin tubes. 5ml of ampicillin and kanamycin selection media are taken in a tube and with the help of a toothpick, we inoculate the cells in the tubes by streaking from our growth plate. The tubes are incubated overnight at 37C in shaker.

The next day, 1-5ml of cultures is centrifuged to make pellet at 12000RPM. This bacterial pellet is re-suspended in to resuspension solution (200ul). Vortex until the mixture is evenly mixed. Lysis solution (200ul) is added to the mixture and mixed immediately by inversion. 350ul of neutralization solution is added to precipitate the cell debris. Pellet the cell debris by centrifuging for 10min.

Meanwhile column is prepared by inserting GenElute Miniprep Binding column into a provided microcentrifuge. Add 500ul of column preparation solution to each miniprep and centrifuge at 12000RPM for 30sec. the lysate solution is transfer to column prepared and centrifuge at 12000rpm for 30sec. discard the flow through liquid. Now add 500ul of optimal wash solution and centrifuge again for 30sec. add 750ul of the diluted wash solution to the column and transfer the colmn in to a fresh collection tube. Finally add elution solution to the column.

2.1.7 Plasmid digestion.

We used NdeI and pst I enymes to check the insertion of insert DNA into the plasmid.

Reaction mixture:

pDNA 3ul
10X NEB buffer 1ul
Water 5.4ul
NdeI 0.3ul
pstI 0.3ul
total 10ul

The reaction mixture is incubated at 37C in the thermostat for 90min.

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