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faint band after plasmid extraction


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7 replies to this topic

#1 Curtis

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Posted 11 June 2013 - 12:14 AM

We asked a company to clone and transform an 18k plasmid for us into TOP10 and last week we received the bacteria in a tube filled with some kind of gel that must be agar. I have never received bacteria like this at room temperature before. So I grew a piece of the gel in broth overnight and extracted plasmid the following day. The plasmid pellet was quite big and I was happy, but when I ran it on the gel the plasmid is really faint. It has a very low concentration. I'm not sure why this happened.

Could this be because this is a long low copy number plasmid? With the shorter one we had no problem before.

The person in charge of our order at the company is really useless and I'm still waiting for his reply about the issue.

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#2 robinlee

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Posted 11 June 2013 - 03:12 AM

I suggest you to pick single clone to amplify the plasmid.

#3 phage434

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Posted 11 June 2013 - 04:50 AM

An 18K plasmid is likely to be in a low copy origin, such as pBelobac or pFOS1. This will give relatively small amounts of plasmid. But I would second the recommendation to be preparing from a single colony.

#4 Curtis

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Posted 11 June 2013 - 08:56 AM

The company sent me the plasmid in a separate tube as well. An induction solution was also included. Not sure how to use that. I really hope I can transform it. Last week we transformed 15k to electro dh10b without any problem...thanks guys.

#5 phage434

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Posted 11 June 2013 - 09:38 AM

There are inducible copy number plasmids -- ones with low copy number until induced, and a higher copy number after induction. Your plasmid may be one of these, in which case you would want to induce it prior to preparing the plasmid.

#6 Curtis

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Posted 11 June 2013 - 10:08 PM

The company just replied that the bacteria are JM108, not TOP10. They made a mistake in the data sheet. Really useless staff. They also forgot to sent me the protocol of induction. Can you tell me the protocol?

#7 phage434

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Posted 12 June 2013 - 04:49 AM

Most likely it would be IPTG, at about 1 mM final. But it also could be arabinose, cumate, or rhamnose. What is the antibiotic resistance? if it is not chloramphenicol, you could also try chloramphenicol amplification.

#8 Curtis

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Posted 12 June 2013 - 07:11 AM

It's kanamycin. I also think the induction solution must be IPTG. It's written 1000x on the tube.




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