dilution cloning


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Presented by, Pratiksha Srivastava M.tech+B.tech(BT) 8th Trimester jv-b/09/1185:

Presented by, Pratiksha Srivastava M.tech+B.tech(BT) 8 th Trimester jv-b/09/1185 DILUTION CLONING

Cell cloning:-:

C ell cloning:- Cell cloning involves production of a population of cells derived from a single cell. Cloning is relatively easy for continuous cell lines. A transformed cells have higher cloning efficiency compared to normal cell. Not hard to distinguish individual colonies. Cloning may be carried by two approaches- Monolayer culture Suspension culture

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Monolayer culture Petri dishes,multiwell plates or flasks can be used for cloning by monolayer culture. It is easy to remove individual colonies of cells from the surface where they are attached. Suspension Culture Cloning can be carried out in suspension by seeding cells into viscous solutions( methocel ) or gel(agar). Daughter cells remain intact and form colonies in suspension.


DILUTION CLONING Dilution cloning was first introduced by Puck and Marcus, 1955 It is the most widely used technique Based on observation that cells diluted below Certain density form discrete colonies(monolayer cells). Trypsinize the cells (at log phase) to produce a single cell suspension. Undertrypsinizing will produces clumps and overtrypsinizing will reduce viability of cells.

Steps involved in dilution cloning:-:

S teps involved in dilution cloning:- Trypsinize the cell to produce single cell suspension. Up to four dilution steps may be necessary to reduce a regular monolayer concentration suitable for cloning. When the cells round up and start to detach, disperse the monolayer in medium containing serum or trypsin inhibitor. Seed the cell into multiwell dishes, Petri dish and culture flask. Then incubate it for 1-3 weeks and after growth isolate the cell.


Isolation of cells- ISOLATION OF CLONES FROM PETRI PLATES If cloning is performed in Petri dishes, there is no physical separation between colonies. This separation must be created by removing the medium and placing a stainless steel or ceramic ring around the colony to be isolated.


STEPS :- 1. Examine the clones, and mark those you wish to isolate with a marker on the underside of the dish. 2. Remove the medium from the dish, and rinse the clones gently with D-PBSA. 3. Using sterile forceps, take one cloning ring, dip it in silicone grease, and press it down on the dish alongside the silicone grease, to spread the grease around the base of the ring.

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4. Place the ring around the desired colony. 5. Add sufficient 0.25% trypsin to fill the hole in the ring (0.1–0.4 mL, depending on the internal diameter of the ring). 6. Close the dish, and incubate it for 15 min at 37◦C. 7. Then isolate the clone with the help of pipette.

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Isolation of monolayer cells

Isolation of cell from multiwell dishes:

I solation of cell from multiwell dishes 1. When growth of cell comes in multiwell dishes , then cells can be isolated by adding trypsin in wells. 2.Trypsinization can be done to produce a single cell suspension. 3. After adding trypsin cells become in suspended form then add media to the flask and when growth comes isolate the desired cell .


ISOLATING CELL COLONIES BY IRRADIATION STEPS:- 1. Select the desired colony, and mark it with a felt-tip pen or a ring marker. 2. Select a piece of lead of appropriate diameter. 3. Take the flask to the radiation source. 4. Invert the flask under the source. 5. Cover the colony with a 2-mm-thick piece of lead. 6. Irradiate the flask. 7. Return the flask to the sterile area. 8. Remove the medium, trypsinize the cells, and allow the cells to reestablish in the same bottle, using the irradiated cells as a feeder layer.



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