01 July 2008
11:23:00 AM
I am flattered by the overwhelming response. But I wish I knew it was all genuine, after all we know the underlying causes of the enthusiasm winks* Nevertheless I appreciate the quries that simply depicts everyone’s inquiry mind. So anyway, the explaination was too long, so I thought maybe I’d address them as a blog post.
Sharon, Andika, Ying Chee, Yvonne, Jean
Transfection is mechanism of the introduction of a foreign material, a plasmid or siRNA into the cell (mainly eukaryotes). Introduction of a plasmid into a cell is also commonly known as infection, like jean mentioned it is using virus as a vector. Introduction of siRNA is what I dealt with. The technique is manual. There are many different types of transfection, calcium phosphate transfection, electroporation transfection, transfection using cationic lipid reagents and transfection using DEAE-Dextran. These are just examples of the different ways the foreign material is being introduced into the cell. In my experiment, we adapted the cationic lipid reagents, using a lipid based reagent to form a complex with the foreign material to enter the cell. More specifically we used Lipofectamine 2000, they say it’s the best cationic lipid reagent.
siRNA is more commonly used as a knockdown, to inhibit expression of a particular gene target. In my experiment, a siRNA (short interfering RNA) binds to the paticular mRNA strand to inhibit the production of a paticular protein. As the siRNA cannot directly bind to the mRNA, we use lipofectamine, a lipid based reagent to react with the siRNA. This forms a complex that will enter the cell by puncturing the lipid bilayer of the membrane, and bring the foreign particle into the cell. siRNA would then bound to another protein to unfold into a single stranded siRNA, one called antisense and another sense. The antisense ss siRNA would find its way to the nucleus to bind to the targeted mRNA to inhibit translation.
Yvonne, Cornelyus
For liquid nitrogen, it is stored in this pretty huge steel container with several racks inside which holds your cell sample in a small tube called, cryovial. As you already know liquid nitrogen is approx. –196 Degress Celsius, which explains the huge steel container. So you just have to take the cryovial out and then thaw it in 37Degrees Celsius water bath. One caution to take note, is to not allow the line of the water to meet the brim of the cryovial, just in case water seeps into the sample. Usually all of the cells in the cryovial are used and it is aliquotted into a culture dish. The amount of media added is respective to the culture dish size, and the type of media added is respective to the cell type. Swirl the dish gently in all directions to help promote the even spreading of the cells before incubating them at 37 Degrees Celsius at 5%CO2 concentration.
Ying Chee
Preparation of buffer may seem like a big deal but it pretty idiot proof with the recipe. In MCT we already had all the necessary and its just adding them all together. Its a little more manual here. My mentor handed me a recepie containing the “raw materials” for Lameli buffer and TBS. Both recepies are for 10X concentration and 1 litre. We use TBS and Lameli buffer all the time, so preparing 10X is a concentrate stock solution. Usually we use 1X, so we'll dilute it, if not we can alter the changes to get whatever concentration we want. He didn’t feel like spoon feeding me on the first day, so he made it a little more difficult by only giving me the molarity of the raw material and the wanted molarity, and made me calculate the mass. Catch no balls? I thought so. Recipe looks something like this.
Preparation of 1L 10X Laemli Running Buffer
0.25M Tris Base (MW 121.1)
1.92M Glycine (MW75.07)
1% SDS (FW288.4)
A recepie is something like above. So I was told to prepare 2L of this buffer. It very much like PIPC, where you’ve gotta calculate the mass of what you want.
Tris Base à 1M = 121.1g
Therefore to get 0.25M,
0.25M = 121.1/ 4
= 30.275g
However I was to prepare 2L, thus
30.275 x 2 = 60.55g
Glycine à 1M = 75.07g
To get 1.92M,
0.02 M = 75.07/50
= 1.5014g
1.92M = 1.5014 x 96
= 144.134g
For 2L,
144.13 x 2 = 288.26gSDS à 1g = 100ml
1000ml = 10g
2L = 20g
which is 1% of 2L
So after all the calculations are done, you just have to add the different raw materials and their respective amounts together, to make your buffer. Top the rest up with DI water and it is always placed on a stirrer to ensure through mixture of the buffer.
LiPing
Lastly, the option of making your own buffers and solutions gives you the benefit of optimizing your experiment conditions. For example, I prepared my own stacking and separation gel for western blot. If you can remember ancient molecular biology, separating gel is the gel that your proteins move across. Thus the composition of the separating gel is critical to obtain your results.
Say if you want to detect a protein in a cell of 12kbs (which is relatively small), you would tweek the composition of the separating gel to eliminate most of the bigger proteins (such as more than 60kbs). Acrylamide is one of the “ingredients” in the recipe, which strictly governs the pore sizes of the gel. Higher percentage of Acrylamide would increase the gel’s tightness and hence smaller pore size allowing only smaller proteins to pass more efficiently. This would then eliminate having bigger proteins in your gel and decreases the incidences of non-specific binding to the primary antibodies later.
I hope this chuck settles all the question marks. Any questions pertaining to this post, please feel free to ask.
Thats all folks.
GLAD
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