Modern Cell Biology Techniques for Medical Research- Poster summary of 2010 summer internship at KU Cancer Center

2. Cultured cells from tissues allow us to create different cell lines to be used in research. For example, as in the mitochondrial studies distinguishing differences between normal and malignant cells

3. These cultures can be preserved in cryo banks or grown in culture cycles, in an incubator, for an indeterminate period of time

4. Polymerase Chain Reaction is used as a method to amplify very small amounts of DNA in cells.

5. The end result should be that thousands to even millions more DNA strands should be replicated and amplified.

6. PCR can clone and amplify specific regions of DNA for further biological research.

7. Agarose Gel Electrophoresis is a type of electrophoresis used to separate DNA and RNA by molecular size and length.

8. Agarose gels are easily cast and used compared to most other forms of gel electrophoresis.

10. It is carried out in a sterile environment (flasks and cell culture hoods) and an incubator to grow cells at 37oC, the normal body temperature of mammals to simulate typical living conditions. (Figure 1).

11. The media contains nutrients necessary to the growth and development needed for selected cell lines.

12. 1% Agarose Gel is most commonly used.

13. To make Agarose Gel, prepare approximately 1L of TBE Buffer solution and 100mL of a 1% Agarose solution

14. Boil the solution and then cool down the solution to about 60 degrees while swirling it so that the bottom of the solution does not solidify.

15. Pour the 60 degree gel it into a gel rack and let gel solidify. Remove the comb carefully.

16. Load the molecular weight ladder in the first well and then load the DNA/RNA

17. Hook the gel rack to a power source

18. When you see that the DNA ladder has reached the bottom of the gel, turn off the power source

19. Finally, put the gel of UV light and the Ethidium Bromide will glow.

20. Extension: The final step in PCR where the temperature is raised to 72 degrees, the polymerase then synthesizes a new strand of DNA using the dNTPs to form complementary strands

21. The DNA growth should be exponential until the polymerase loses activity because the sources of dNTPs and primers become more limited

23. When pipeting try not to touch the bottom of the flask, if touched the cells residing there could be disturbed.

24. Cell medium needs to be changed periodically whenever the serum medium and other vital components are depleted

26. Figure 2 shows the reaction

27. A PCR machine is used to denature and anneal the DNA.

28. The materials needed to perform PCR are the DNA Template, Forward and Reverse Primers, Taq Polymerase, a buffer solution, dNTPs, and the PCR Machine

29. PCR consists of 20-40 repeated cycles in which three major steps occur:

30. Denaturation: The first step of PCR where the DNA template is heated to 95 degrees causing the hydrogen bonds in the DNA to melt leaving single stranded DNA

32. Add new media and suspend the cells in it, then place the medium in the flask

33. Keep it in an incubator or a growth chamber that simulates the temperature of an ideal environment for the cells.

35. Supplies needed are TBE (Tris/Borate/ EDTA), Ethidium Bromide, a gel rack, a comb, a power supply, loading dye, and molecular weight markersReferences http://www.celprogen.com/store/index.php?cPath=26_44_46_59_61 http://www.wisbiomed.com/ec/index.php?cPath=12&main_page=index http://www.statemaster.com/encyclopedia/Gel-electrophoresis.