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Is my insert present???


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#16 HomeBrew

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Posted 30 October 2009 - 04:19 AM

I agree with almost a doctor -- it's not time to despair or start questioning life choices yet...

I also agree that there are a lot of steps where a cloning experiment can go wrong and that there are a lot of people here in the BioForum with experience and tips that can help you get your clones. The more detail we have about how you proceeded, the more we can help. For example, I asked you what the nature of your transformation was, because there are lots of ways to transform bacteria -- chemical competency, electroporation, conjugation, transduction -- and I didn't know which method you used.

So, from what you've said, it appears you used chemically competent cells for transformation, and that the cells were reasonably competent, depending on how much you plated to arrive at 60 or so colonies. It's not clear how you screened these colonies -- how did you arrive at the one clone you're testing?

The presence of colonies on the no ligase and mock controls is a bit troubling, but that's why we do controls. For example, the colonies on the no ligation control might indicate that your vector prep was incompletely digested. My first tip for you regarding this is to always, always, always gel purify your vector and insert digestion preps before using them in a ligation. Additionally, in my lab we always add a "sunblock" to agarose gels from which we are going to excise fragments, to protect the DNA from UV damage. This is quite simple, and increases our success rate dramatically -- simply add 1 mM guanosine (e.g. Sigma G6752) to the 1x TAE used to cast the gel and to the 1x TAE running buffer used during electrophoresis (see Grundemann, D., and E. Schomig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. Biotechniques 21(5):898-903. There's a pdf here). I would rank gel-purifying your vector and insert digestions and using a UV protectant as "critical steps" to success. Of course, the way you do the digestions also matters -- what digestion protocol did you use? What company did you get the enzymes from?

Now to the ligation itself. How did you perform it? What ligase did you use, and at what temperature and for what duration? Was the ligation buffer fresh (the ATP in the buffer will break down on repeated freeze-thaw cycles)? Although personally I don't usually find it critically important, what ratio of insert to vector did you use? We have switched to using Ready-to-go T4 ligase exclusively, as it avoids many of the problems associated with ligation failures.

On the competent cells you used -- did you prepare them yourself, or were they commercially acquired? If you prepared them yourself, what protocol did you use? In my lab, we find the rubidium chloride method to work best.

As you can see, a cloning experiment can go horribly wrong at any step, and introducing little tweaks based on experience can dramatically effect your chances of success. Because of the tweaks we've added over the years to our standard protocol (I've been doing this for twenty years), cloning in my lab has become rather routine; it's rare that we don't get the clone we're looking for on the first try.

If you're willing to provide us with detailed information about your procedures -- include everything, we'd rather have information we don't need rather than the other way around -- we BioForumers can help you tweak your protocols to maximize your chances at success.

#17 Allint

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Posted 30 October 2009 - 03:24 PM

I'll just add my question about checking the transformants to this topic.

I'm new to cloning. I'm trying to clone a 750 bp insert into a 5.5 kb vector. I did the double digestion (BamHI and ClaI), ligation, transformation and miniprep from a couple of transformant colonies.

When I do a "diagnostic" restriction of the plasmid, using BamHI and ClaI, I get a band that is in the right place on the gel (cca 750 bp). It's possible to get that band size even if I have multiple inserts per vector or concatamers, right? So, how can I tell the difference between these three products using only restriction analysis?

#18 HomeBrew

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Posted 30 October 2009 - 04:57 PM

I'll just add my question about checking the transformants to this topic.

I'm new to cloning. I'm trying to clone a 750 bp insert into a 5.5 kb vector. I did the double digestion (BamHI and ClaI), ligation, transformation and miniprep from a couple of transformant colonies.

When I do a "diagnostic" restriction of the plasmid, using BamHI and ClaI, I get a band that is in the right place on the gel (cca 750 bp). It's possible to get that band size even if I have multiple inserts per vector or concatamers, right? So, how can I tell the difference between these three products using only restriction analysis?


Cut with one enzyme and see if produces a single band of about 6.3 kb.




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