Analyzing a Ligation before Transformation


I am afraid if I fail again! Oh, no! It is too bad. I did it until I cut the band from the agarose gel..but I did not do DNA isolation perfectly. Last night before, I was very tired and sleepy so that I did not see the undissolved gel! That was terrible thing I could do. I cut the gel and dissolved it with sodium iodide, incubated 37C with vigorous vortexing, even until 10 minutes. I was surprised yesterday morning when I took the tube from the cool room and then spinned it, yeah, I saw the undissolved gel! I did wanted to throw away the tube and cry..but still, I kept those tubes and added more NaI, incubated again at 4C for several hours and then continued to isolate the DNA. 

After I measured the DNA concentration by spot method, I was pretty sure of the pcDNA fragment, it remained there. But I the PGV-B fragment was none, zero concentration I guessed. Just liked the last experiment I did, I continued to ligate the fragments…and did transformation. This morning I picked 2 clones up and transferred into Amp-LB-liquid. I did not have any idea except to wait until tomorrow, after I do miniprep DNA isolation and check the DNA, until I found a good idea from the internet. Yeah, it is a good idea to check the ligation before doing transformation. We also can observe the result more quickly. We have to sacrifice our sample, and of course, agarose gel (1 g of agarose is 11 Yen), but still it is a good idea.

Now, I am just waiting how it works and think how to prepare the next experiment. I can not keep on focus because I have to prepare for my progress report too.

_______

Postby jamiejlg on 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?

Postby blcr11 on 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.

Postby david23 on 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.

source:http://www.biology-online.org/biology-forum/about10721.html

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