Some difficulties associated with the peg methods are

 

Some difficulties associated with the peg methods are

Ø  Transformation efficiency associated with this procedure is very low.

Ø  There is a difficulty in order to obtain the maximum amount of the protoplast.

Ø  Despite of these difficulties this method is simple and material used in this process is very simple.

Ø  This is the strong reason that peg method is used commonly.

Ø  The transformation of the genomic DNA into the genomic specie has been applied to many fungus species.

Ø  The main three steps that are involved in the PEG are as followed.

Ø  Preparation of protoplast

Ø  Exposure of DNA to PeG

Ø  Regeneration of protoplast

We will degrading enzymes can be used for the germination of the species. Lytic enzymes which were used for the degradation there ability to degrade the cell wall is different so the testing efficiency of the proto plast is different. The dilation is always or mostly performed in the presence of the of buffer or the greater salt concentration.

In order to take DNA with the help of protoplast the incubation of protoplast was required in addition to peg and another incubation period is required. In order to trap the dna in the fungal cells protoplast fusion is performed. As peg is involved in up taking of the dna. But its greater role is unknown because only the fusion of the protoplast is not required in the uptake of the DNA. AFTER THE treat ment of peg with protoplast its washing is laso required then it is regenerated and replace on the selective medium. Another fungus s. nodicum can also transformed by the hygromycin and also fungus has also been successfully transformed. Another type of fungs can also be transformed and causes the the disease to the wheat crop. It can be transformed so it can not causes harm to the wheat crop.

All the instruments used in this process can be subjected to the sterilization process by using the autoclave. This condition is applied in auto clave 15 ps temperature 120 degree c temperature for 15 min.  in case of liquid material that are being used are can be filtered.  For the preparation of all the solutions water is used it should be ultra pure.

The solution used in this procedure must be prepared one day before its use.

Ø  Take about 600 ml of water in the baker.

Ø  After that the material which is used in this is weighted before dissolving in the it.

Ø  The material that are being added in water are titrated nitrate sulphate yeast and sucrose is used.

Ø  After the addition of this material then rises the level of water up to lL

Ø  PH adjustment is not required solution is added in two different size flasks.

Ø  After that the solution is sterilized by autoclave and stored at the room temperature

Ø  Take about 500 ML water in the baker and add cacle kcl and water are added in it.

Ø  When these materials are added in the water then the level of water are raised up to 1L.

Ø  Add this solution into two bottles and then sterilized it by autoclave and stored it at room temperature

Ø  This solution is must be prepared freashly and then buffer solution is measured.

Ø  Stair this solution and add it to the centrifuge tubes.

Ø   These tubes then can be centrifuged for the duration of about 10 minutes.

Ø  About 400 ml of the water is added into the beaker and then washed it by the mycelia and protoplast.

Ø  All the beakers that are than made these are covered up by the aluminum foil.

Ø  And then sterilized these bakers by using the different techniques of the autoclave at standard level.

Ø  Add 100 ml water in the baker then add yeast sucrose and tryptone in this tube of the water.

Ø  After mixing this solution rise the level of that solution of water up to 200 ml.

Ø  Then again same setup like as sterilization and autoclaving the solution.

Ø  After centrifugation suspended protoplasts and then again resuspended the protoplast.

Ø   Then incubate these tubes for four hours.

Ø  When suspension is obtained mixes it and then places it in tubes.

Ø  Then add the hygromycin b and then put this solution in the petridishes..

Ø  Then incubate the dishes at different temperature.

Ø  Again incubate these dishes at the temperature of the 30 degree c.

Ø  Then incubation of these dishes will be performed and the colonies will become visible.

Ø  Then again incubate the dishes again at the temperature of 30 degree c.

Ø  With the help of scalpel cut a piece of the colony and add the hygromycin B.

Ø  Incubate the plants at the temperature of 25 degree centigrade. And those transformants are healthy.

Ø  For the isolation of the species use the tip of the niddle.

Ø  Again then incubate the plants to obtain the new colonies.

Ø  In order to grow fungi in large amounts than fused medium is required in the small amount.

Ø  When different amount of polyethylene glycol is used then there are variation in efficiencies and results.

Ø  If we can store hygromycin properly than we can use it for the five years.

Ø  When peg solution is added then the solution become cloudy. This is because of joining of protoplast.

Ø  In case of fungi the process for separation is different then takes the help from the taken results.

Ø   If we add antibiotics in the control pallets then the growth of protoplast is reduced so do not add the antibiotics to allow the repaid growth of antibiotics..

Ø  When incubation is performed then there is need to performed the tap tubes for many times.

Ø  In case of fresh medium as antibiotics are absent so there are chances that this medium can be contaminated.

Ø  By the bacteria so check the culture to avoid the contamination of the medium.