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analyzing a ligation before transformation

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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analyzing a ligation before transformation

Postby jamiejlg » Thu May 10, 2007 7:20 pm

is it standard practice to run a ligation reaction on an agarose gel to determine if the pieces were ligated before proceeding to transformation?
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Postby blcr11 » Thu May 10, 2007 7:53 pm

It used to be that you'd do the ligation in 20 ul and sacrifice 5 ul for an agarose gel. You can also linearize the putative product and compare the size of the ligation reaction to linearized vector alone-or look for a fragment of the correct size if you're cutting on both sides of the insert--but that requires using enough DNA to see the (usually) smaller piece of DNA. Linearizing the construct is a little more sensitive than trying to compare ligated vector + insert to vector alone plasmid DNA. You can also just go ahead and screen for inserts later, but of course, you'll waste a day or so if the ligation didn't work at all. Pay me now, or pay me later, I guess, with respect to time utilization.
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Postby david23 » Fri May 11, 2007 4:01 pm

yeah when I do it, we are usually required to.

After transformation, the incubation and growing of the bacteria takes time right? You dont want to end up with nothing.
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Postby canalon » Fri May 11, 2007 8:43 pm

Sometimes you also assume that it works, and hope that you will get something without bothering to check, and you will sort your clones in the end. Never had a ligation finishing too late?

And sometimes you simply cannot check, just because it is impossible to know what to expect. For example when you are trying to clane the fragments amplified after substractive hybridization (note that there is probably many other situation where this happens, but this one happened to me recently...)
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Serious Ligation issues :( (Part science part rant)

Postby dinofernando » Fri Aug 31, 2007 2:46 am

Hi everyone,

Well i've started my ligations and now that i'm on my third attempt to transform my XL1 supercompetent cells I've come for HELP!!!

The last two that I did were directly from a .7% nusieve gel that i washed with dH20 spun and removed supernatant (well 120uL from the top since no layer was seen). I set up my ligation reactions
1. plasmid 2. Insert 3. I&P

The first time the ligation ran overnight, the second time it ran for about ~26hrs. I added my LB shake for 1hr at 37C then spin remove some supernatant and resuspend my cells.

Plate on Kan-LB plates and left overnight for about 15hrs. Big problem was I squirted my transformed cells onto the lid of my plate by accident, so I grabbed as much from the glass spreader and spread on the agar :oops: :shock: I have not thrown the plates away they are still "growing" now and i'm doing a third ligation now because their is nothing on them.

This third time I have used a kit to purify the gel extract that I have ended with 50uL of insert and plasmid. And my ligations are running in a 10uL volume. They have a formula to determine what volume to use, the recommend a 3:1 insert to vector ratio. I did that and now waiting (they recommend a 3hr. room temp ligation time).

Now for the question. Is there anyting I can do besides running a nusieve to check my ligation. I don't think I have enough volume to even run a gel !!!
I"m so frustrated...........I think the second time I used less Kanomicin since I did my dilution wrong when making up my plates and used less. But still no colonies on any of my plates. I ran 1) Plasmid 2) Plasmid +insert 3) Insert 4) Cells only. The plates on both occassions are clear as the time they were poured except for condensation on the lids. :oops: :(

At this point i'm not sure if its my technique or the prod i'm using. My PCR worked wonderfully and I got nice clean bands for my vector and insert. Then I did a DNA gel extraction then the clean up etc.

If any has any suggestions or kind words I'd really appreciate it. I'm feeling very very "unconfident".

bahhhhhhhhhh
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Postby blcr11 » Fri Aug 31, 2007 4:43 pm

Is your ligase dead maybe? Do you have a positive control to show that the enzyme works? I don't see there's much else you can do but proceed to the transfection if you don't have enough sample to run on a gel.
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Postby dinofernando » Sat Sep 01, 2007 1:32 am

I think the ligase should be ok. Its used commonly among all in the lab and they seem to be using it fine.

If this third try doesn't work I'll make up enough so after ligation i can run on a gel.
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Postby dinofernando » Sun Sep 02, 2007 2:26 pm

Well I checked out my transformants over the weekend and THEY GREW!!! But I also got growth on my controls however not as much. The colonies on my transformed plate are few and "thick". The controls are thin and many so I don't know which is good or bad???

They have been in the incubatior since friday and its now Sunday. I"m a bit worried to keep them in the incubator since I won't be able to do a PCR screen till tuesday.

Should I strore them in the fridge or can they continue in the incubator? that would make it more than 72 hours at 37C......

ANy opinions?
Thanks!
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Postby ShineGene » Sun Sep 23, 2007 2:17 am

You had better add PEG to ligation buffer.
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