LANT VIROLOGY LAB TRAINING REPORT
Amplification of TuMV-VPg gene
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
|H2O : 33.5ul|
|5X buffer : 10ul|
|10mM dNTP : 1ul|
|Primer F : 2ul|
|Primer R : 2ul|
|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|
|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.
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.
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 :
|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.
|10X NEB buffer||1ul|
The reaction mixture is incubated at 37C in the thermostat for 90min.