Is my insert present??? - (Oct/28/2009 )

It's an interesting problem, rocketfan86 -- let us know how it progresses towards a solution...

BTW, welcome to the BioForums!

HomeBrew on Oct 29 2009, 03:37 PM said:

Thank you HomeBrew!

So i ran PCR on 1% Agarose Gel and the positive control (MSH3/pCR4-TOPO) worked but the construct did not. I need to figure out how to make it work.

Right now, i will run another gel to look at another digestion of MSH3/pCS2MT. I have used another restriction site (BamHI) and an original XhoI to see if the insert is present so the size of the insert should be ~3.7kb. If that doesn't work, i guess i can sequentially digest EcoRI and XhoI to see if that makes a difference.

Keep you posted.

Let's back up a bit... What was the nature of your transformation? Did you get a lot of colonies, or only a few?

At some point, it makes more sense to just start over and re-clone it.

HomeBrew on Oct 29 2009, 04:59 PM said:

Nature? Just thawing DH5-alpha E. coli on ice , put the ligated plasmid/insert into the bacteria, flick tube, rest on ice for 15 minutes, heat shock for 30 seconds at 42C, put back on ice for 2 then plate. It was getting late so i skipped the adding of SOC media and incubating in 37C shaker to recover.

I got about 60 colonies. + ligation control got about 23 colonies but - ligation control got 20 colonies and -/- ligation/transformation got 12 colonies.

Makes me think there is contamination or incomplete digestion.

Probably need to make some new Amp+LB plates.

For this construct it took me 9 times to finally get one colony that looked like it contained the insert...i guess i'm losing hope fast.

Trying the other double digestion with another enzyme and XhoI showed a band the size of an empty vector. Needless to say I was devistated. To top that off, the other construct i've been trying to make, EXO1 and pFLAG-CMV2, once again showed only empty vector. Very frustrating indeed and still wondering i i decided to be a graduate student.

Hi rocketfan86, dont despair just yet. Science (and cloning in this case) can be a tricky matter but is rewarding in the end.

From reading all the posts I understand you are trying to prove a particular mini-prep contains your insert. In my opinion I think you are getting stuck in the wrong stage, I totally agree with homebrew that sometimes starting all over is better, it might save you more time than you initially imagine.

In one of the latests posts you said you have 22 colonies in you ligation, but 20 in the negative control... it looks to me like the problem is your ligation then and you are trying to prove your positive is positive when in fact is empty vector (quite frankly I think you have enough proof to say is a fail, and is just empty vector).

Could you post your full protocol: how do you obtain the insert? how do you prepare your vector? what's your ligation protocol? what's your transformation protocol? I think we would be able to help you much better if you tell us all this information and we can walk thru the problem step by step.
Dont take this the wrong way, but if your other construct (EXO1 and pFLAG-CMV2) isn't working better, you might be missing some point and we might be able to help define it.

Hope this helps.

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.

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?

Allint on Oct 30 2009, 07:24 PM said:

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