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DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Hello to All - DNA Techniques

Hello to All - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #1  
Old 09-18-2006, 01:55 PM
Pipette Filler
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Question Hello to All



Dear All,

I am a graduate student doing Masters in Industrial Chemistry majoring in Biotechnology. Currently my work deals a lot with genetics. Plasmid isolation is what I am doing now. I have very little knowledege in this and therefore I have to catch up as soon as possible on the theoretical part of my work.

I have done the isolation of the plasmids and run the agarose gel electrophoresis. The first time was a total failure as even the DNA ladder was not showing anything under the UV illuminator.

The second trial was partly a success as the plasmid for pUC19 was visible on the gel without digestion. The result shown that it was about 1.5kb. However, according to the source, pUC19 was supposed to yield plasmids as big as 3.1kb. Why is it that I only obtained results at 1.5kb?

My strain did not show any plasmids at all. According to literature, the size should be about 23kb. I used Kady and Liu method for plasmid extraction. What could have happened?

Thanks.
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  #2  
Old 09-19-2006, 05:26 AM
oBWhat's Avatar
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Default Re: Hello to All

Hey there hsip_15!

Now I am not sure what you are doing. Are you transforming E.coli bacteria with PUC19? If so, you should be getting a 2.7 kB size plasmid if you are using the puc19 from any company as they should be the same size.

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"pUC19 is a commonly used plasmid cloning vector in E.coli. The molecule is a small double-stranded circle, 2686 base pairs in length, and has a high copy number. pUC19 carries a 54 base-pair multiple cloning site polylinker that contains unique sites for 13 different hexanucleotide-specific restriction endonucleases"

Now, either the first time you had an ethidium bromide staining problem or your DNA/ladder was lost before / during loading...

At this point, anything on your gel is good!

What is "your strain"? at 23kb, it is a very big plasmid! you may be losing some during purification. are you using columns or other method?
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Old 09-19-2006, 02:15 PM
Pipette Filler
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Talking Re: Hello to All

Hey..Thanks alot for the reply. I se high hopes when I get at least a reply. Thanks.

Well, that is pUC19 that I get from another student's stock.

Then, thanks.. the gel did show something and that was great. The ladder shown too.

The 23kb plasmid is a metal reductase coding plasmid..if I am not mistaken about what I am saying. I will be extracting again with the Kado and Liu method. And then, I will be cutting it with two kinds of enzymes Pst and Hind. This will cut my plasmids into smaller fragments so that it will show on the gel instead of stucking to the wells due to the large size. I will have to analyse the plasmids if I get any..

Do you have any services like sharing of journals or resources here? Some of the journals I cannot get in my library and also online especially when they are not for free.

Thanks.
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