Science Buddies: "Ask an Expert"

I have have a way to extract the DNA from E.coli, primers to amplifythe specific gene I want, and a forming idea of how to insert it back into the E. coli using vectors, I have came accross another question that is making me run around in cirles. Before I allow the amplified DNA to insert themselves into the vectors, do I need to separate the amplified DNA from the reation mixture? I thought so, but then I second guessed it, and now I am confused. If so, how do I do that?

Thank you,
mmb11

Greetings,

It's good to see that you've made quite a bit of progress with your project.

I'm assuming that you found a PCR machine. Will you be able to perform gel electrophoresis as well? Or are you going to use one of the methods suggested by Donna Hardy?

If you can perform gel electorphoresis, the easiest thing is to gel purify your PCR product. This ensures that you'll get only the product that you want and eliminates the other things in the PCR reaction.

If you don't have access to gel electrophoresis, please let us know and we can post other alternatives. They exist, but they're not as simple and cutting out your specific band and gel purifying the product.

It's great that you realized that you need to separate the reaction mixture from your PCR product. The problem is that some PCR buffers may contain EDTA (or TE), and this can interfere with with the ligation reaction. So even if you can't gel purify the product, post here and we'll help you come up with a way to separate your PCR product from the other components in the reaction.

I hope this was helpful. If you need clarification, please let me know.

Best of luck,
Linh

Hi mmb11,

You will probably obtain better results if you remove the smaller molecules from the amplified DNA sample. This is usually done with small spin column containing gel filtration media that separate DNA products by size. The amplified DNA is a larger molecule so will elute through the column and the unincorporated nucleotides will be retained in the column because they are smaller.

A number of companies sell spin columns, but we can explain how to do this if you have access to any type of gel filtration media and some small chromatography columns. It sounds like you have access to a laboratory with PCR equipment, so ask if you can "borrow" a couple of these spin columns.

If you can't do this step, go ahead with the experiment because it probably work, but it will definitely be more efficient if can clean up your sample.

Let us know if you need help locating materials for this step of your experiment.

Donna Hardy

http://www2.epochbiolabs.com:563/pcrcle ... e=products

If this looks appropriate to you, I think I will just buy this to use. If so, what are the sizes measuring? It says 50 preps or 250 preps. Preps meaning what exactly?

Thank you
mmb11

Hi mmb,

Looks like things are going well with the planning.

The link you gave for the columns is the kind of thing Donna was talking about. That company also has the ones for extracting from a band from a gel /gel extraction columns (also listed under DNA cleanup). 50 preps means cleans up 50 standard sized PCR reactions. 50 preps for the gel extraction may be less than 50 bands because you need to weight the band and may need to divide it between more than one column. All that stuff is in the instructions for the columns. And all the buffers come with the columns.

Did you locate some gel running equipment? It really is the best way to tell if your product is the right size or not, or if you have to tinker with the PCR conditions. Then when you have the conditions right I usually scale up and run a few PCR reactions to make sure i have plenty of product for cloning into the vector because you lose some product at each stage.

good luck,

Caroline

Hi mbb,

Yes, these are the columns I was talking about. 50 and 250 preps means there are either 50 or 250 columns per package. You probably won't need 50 columns, so I suggest that you call the technical support department of this company (there are several companies that sell columns like this) and tell them you are a high school student ask them for a sample of a few columns. You should ask the technical representative to e-mail you the instruction manual for the product, or download it from their website. These columns are designed to be run in a centrifuge, but you can run them by gravity if you don't have a centrifuge. Running by gravity will result in a more dilute sample, however, so it is better to use a centrifuge. The columns can be reused, if necessary, by putting 2-3 ml of buffer over the column to rinse out the nucleotides and other small molecules.

You are doing great on your background research. Let us know if you have any other questions.

Donna Hardy

Caroline

Can you explain a little more what you mean by gel running equipment?

Is this a gel electrophoresis equipment?

Thank You,
mmb11

sorry. gel running equipment is the same gel electrophoresis equipment. For the kind of experiment to check PCR band size and purify it, this would be a horizontal electrophoresis using an agarose gel.
the lab that has the PCR machine will almost certainly have one of these you could borrow too.
-Caroline