How to ligate ds-oligos into a vector? - (Aug/25/2004 )

Hi zhgljj,

check this post http://www.protocol-online.org/forums/inde...?showtopic=6527

you can run a gel after annealing along side with single stranded oligos. You will find ds-oligos migrate differently from ss-oligos.

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Hi! I am facing a problem with a 50 bp single strand DNA as you do. If I undesrtood your message, you synthesized the first and secong strand and alow them to annealing in the bench. After that you ligate in a vector ( I think you have to phsophorilate and diphosphorilate them). I am right about the annealing? Thank you! Angela

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Hi there,

I have been reading the discussion and find it very interesting. I am myself cloning a 87 bp oligo(ordered from sigma). I have done the following.
I have taken 2 microgram of each of the oligos in 2XSSC in a total volume of 50 microlitre heated it at 95 degree celsius for 15 min then let it cool down to room temperature. When I loaded in gel (agarose) I could see a single bright band.By the way I plan to clone the oligo in pGEMT easy after A tail by taq pol.

Now my questions are the following.
Since I see a single band only, do I still need to purify it.
If I need to purify can I use Qiagen gel extraction kit? or shall I try to purify it from LMP? or purification from PAGE is necessary.

My next question is which is better- purification after annealing or before annealing?

mili

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hey, am new.but find this discussion very useful.i have a 80bp oligo that i want to anneal and then use for ligation.the oligos are phosphorylated but i forgot to ask the company to PAGE purify them.Can i just purify the ds-oligo after annealing using agarose gels??
would be great if i cld get soem help

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I think you should purify them before annealing otherwise you may anneal long and short primers together as well as long and long primers together.
It may be more difficult to see this difference on gel after annealing.

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The ologos are not phosphorylated. Therefore, first perform kinasing and then cloning.

Regards

Javedz
www.dnati.com

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hi, Iam also intrested to post my experiences here. since last month, I performed cloning with short ds oligos. totally i did cloning for four ds oligos, they are 54 bp and one is 40bp only, we ordered for sysnthetic ds oligos. Just I have used 3:1 and 5:1 ratio., i got few colonies from both the ratios. yes, i used ligae from different companies for different oligos. it doesnt matter, it works easly with any ligasse. But be sure to have your plasmid digestion. I did with double digestion. when you r doing doublwdigestion, better to precipitate DNA in between two enzymes digestion, so that you will not any error at the end.
hope you will get some information from my epxt.
bye
Nari

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I wanted to ligate a dsDNA oligo into the MCS of my vector (double-digested with NotI and EcoRI). Everyone here seems to recommend dephosphorylating the vector and phosphorylating the oligos for this purpose, even if the vector is double-digested.

However, last time I did this, I was afraid that I could get concatamerisation. In other words, instead of one linker ligating in with an {Eco->Not} orientation, I could get multiple copies going in as an odd number of inverted repeats ie. {Eco->Not:Not->Eco:Eco->Not}. Has anyone else ever had this happen to them? Or is it rare?

Anyway, so to avoid this, I didn't phosphorylate my oligos. So I couldn't dephosphorylate the cut vector. I used a large excess of linker: ~1 uM in 10 ul ligation reaction, with ~40ng of gel-purified, cut vector (4kb). It worked quite well, in fact, most tested colonies were positive. I guess it all hinges on the efficiency of the vector digestion. I think most of the linear DNA that I gel-purified was digested with both enzymes, however it is also possible that the high concentration of linker might anneal to the vector ends, and thus block the self-ligation of incompletely digested vector.

I'm about to try it again with a small linker, I'll let you know whether I can replicate this.

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Update: this strategy worked again with a different linker, 8 of 15 colonies tested were recombinant and positive for the linker. But I only got about 30-40 colonies after transforming 0.5 ul of ligation reaction (described above).

So, you may not need to phosphorylate the linkers or dephosphorylate the vector if you are doing a double-digest on your vector and the digestion is efficient.

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