14 replies to this topic

[Maddie]

Hello everyone,

I'm about to start a ligation to create a library for next generation sequencing. I have zero experience with ligation and other enzymatic stuff, so please forgive me for this amateur question.
I read a post last week about how to set up a ligation. Someone was explaining that it was best to mix product A and product B before adding product C. I can't find that message anymore and do not remember what A, B and C were. What is best to mix the DNA with the ligase and then add the adaptors? or adaptors + DNA and then ligase or adaptors + ligase and then DNA? The person said it was important to avoid..a certain issue I also forgot   . I just remember: Oh this is great info, I'll do that when I prepare my library and then...pfiiut...gone in the wind. Please, help. I use very bad (degraded DNA) for the library prep and it doesn't ligate well at all, so anything that can help, even a detail like that would be greatly appreciated.

[pito]

I always add the enzymes at end..

I start with big amounts and finish with the smaller amounts and in the end I add the enzyme.

But you are speaking about creating a library, but you only speak about adding the adaptors? Are you now going to ligate the adaptors and maybe next week create the library (adding the DNA pieces with the adaptors in the "host" or?)

and about avoiding? Maybe to avoid centrifuging?
Make sure to mix everything? Avoid having your enzyme to heat to much (when adding it, keep the bottle with enzym on ice, and also your samples) ?

[Maddie]

Pito!! It's so nice to hear from you   . It's been a while.

I use extracted DNA (from bones) for the library prep. Following the Illumina protocol, first I do an end repair, I clean-up the reaction with Quiaquick or MinElute, then I add As to the 3'end of the DNA, clean-up and finally I ligate the adaptors to my ds DNA. I've already made a few libraries and when I use PCR product instead of my degraded DNA as a template, of course it rocks. With my old DNA..it's not so nice and when I try to amplify with primers that cover the adaptors, the amplification is really weak.
For the ligation step, I am supposed to mix 10ul of ds DNA sample + 25ul ligase, 10ul adaptors and 5ul ligase. I was planning on making a master mix with ligase buffer, ligase and adaptors and then mix with my DNA but I wonder if it will increase the chances of having adaptors ligating to each other. Should I do a master mix for the ligase and ligase buffer only? Mix DNA and adaptors and then mix with the master mix? Do you think it will make a difference? I do all on ice BTW.

[pito]

Quote

I am supposed to mix 10ul of ds DNA sample + 25ul ligase, 10ul adaptors and 5ul ligase.

I do not get it? You mean 25µl ligase buffer? and 5µl ligase (the enzyme)?

anothering thing: you use µl to classify how much DNA you use and how much adaptors... how much in ng is it? Because the volume doenst mean a lot...

[Maddie]

you're right. Althought in my case, the amount..well..I'm always far under 5ug. I think in this case I may have managed to reach 800ng (if I can rely on picogreen).
Yes 25ul ligase buffer and 5ul of enzyme. Gosh I need a vacation. I'm lucky, today it's snowing so I'm home.

[pito]

Snowing? from where are you ? USA I remember, but where?

800ng is not a lot. But the problem with ligations is that its very tricky.. it all depends on the ratio and a general formula for example is: # ng insert = #bp insert/#bp vector X 6 X #ng vector (bp=basepairs).

this formula is for the calculation of the insert (after adding the adaptors).

And I used a ratio of 6 , the ratio is very imporatant but changes depending on what bacterium you use .. its all trail and error. Very important is that your DNA is clean. This can cause many problems.
Most often they try 1 to 1, 1 to 3 and 3 to 1 , but I preffer using a ratio that favors the insert (use more insert then vector).


Now for tha adaptors: I dont know any formula or ratios by heart, ist there more information in the adaptors itself?
But you can offcourse use the same formula I gave for the ligation reaction itself since adding the adaptors is a ligation too.

(check http://www.promega.c...math/calc06.htm, so you can use the formula on a website...)

And the 800ng isnt bad.. I used ligation reactions with 200 ng of vector and about 300to 350à400 ng insert

Edited by pito, 26 January 2011 - 10:45 AM.

[Maddie]

I don't use vectors or bacteria. I just try to attach short adapters and then the adaptors will hybridize with a complementary sequence on the Illumina platform and the DNA will be amplified and sequenced (see the link). So my stuff is really a very simple ligation. For the ratios they say to use 10ul when you start with 5ug of DNA, or keep a ratio 10:1. 1ul for 500pg. Well, we'll see. I couldn't amplify today and probably not tomorrow either because of the snow. I'm on the East Coast, close to Washington DC. Bad bad weather this year again. Looks like last year. How's it in Belgium?

http://seqanswers.co...thread.php?t=21

[pito]

Oh ok, lol, I am not that awake , I for some reason missed the Illumina protocol thingie lol or ignored the fact you want to fully sequence eukaryotic dna. I really need to pay more attention in the future.

1 to 10 is indeed used ,  I remember that we used that ratio too, but havent used that protocol that often.
But you do mean: when starting with 5µg of DNA, add 10 µl of adaptors? or? And to be honest: I dont get the idea of using µg and µl , a µl means nothing if you dont know whats in it... I mean: 1µl for 500pg ... what does that mean? you mean taking 1µl of adaptors for 500pg of DNA? If thats the case, I do not see the 1to10 ratio...500pg is only 0,5ng..? So you are not using the 1to 10 ratio really.. since you have 5µg of dna...
Or am I again sleeping here and missing the whole idea?

But to be honest: it all depends on the dna you have. Someone here once used a ratio of 1 to 20.. he never got results and in a rage he just added twice the amount and he got a result ... The purity of your DNA is also very important.
Anyway, you might want to check this paper: Improved Protocols for the Illumina Genome Analyzer Sequencing System (http://onlinelibrary...802s62/abstract)


Ah, Washington DC, thats where the white house is , right? They have good universities or colleges there?
For some reason all I can think about, when I think about visiting the usa, is to visit california and lay down on the beach. I think I saw way too many movies showing the sunny beaches of california. (arent there a lot of "old" (retired) people living there? For some reason in the movies they only show the very sparkling and hip , young, california:p)
Its good here: 10°C degrees in the winter.. lol. Whe had a lot of snow and cold tempratures from the start of december untill the end of december (white christmas) but after christmas, start of january it became hotter and hotter..

[Maddie]

Ahhh not fair for the weather.
Here it was so bad last night that people were stuck on the higway, ran out of gas and had to leave their car and walk to the nearest gas station which was..closed because of power outage.

Sorry about talking in volume instead of concentration but I use a kit and of course, I don't know what's in it so I can only follow instructions. They tell us to use 10ul when starting with 5ug of DNA or 1ul if starting with 500pg. Plus, since I know almost nothing about ligations..
I'm trying to get a sense of little tricks I could do that would help a bit. These kits have been designed to work with lots of high quality, high molecular weight DNA.
I can't open that link you sent but it looks interesting. Would you be able to send it or temporarily attach it? Pretty please?

California..ahh yes quite different from here. The retirees go a lot to Florida actually. DC is great because there are many maaaany biotech companies in Maryland and Virginia. Quiagen is here, we have some Aplied Biosystems labs too. Celera is here and many others. We are close to NIH, NIST, so basically we have lots of opportunities to talk and or collaborate with other labs. This is a great place to live for a molecular biologist.
La vie est belle, lalala.

[pito]

The article could really help you I think.

But I can not send it to you. I do not have a pdf file and for some reason I am not able to make contact with the university server... Maybe you need to make a "request article" topic ? Someone else can give it to you then.

I see, a nice place to be .. Are you working for such a company? or at a uversity?

Yes, the protocols are for clean material...

Oh, Ok I understand what you are saying now, its just a fixed volume that has to be added.
You did clean up your dna as much as possible I am assuming? Extra ethanolprecipitation step etc? No vigorous shaking when doing the cleaning steps etc..?

Does the kit mention how much adaptors are in each µl?  I havent used it often, so cant remember anything really about numbers...

[Maddie]

I found out the "trick" I was looking for. It wasn't on the forum but on a protocol I have .
I thought I'd share. It's about a ligation between genomic DNA and adaptors in the presence of PEG. Since PEG is highly viscous, the authors recommend to vortex the master mix before adding the ligase and then mix gently after adding it. Didn't know ligase was fragile.

[Maddie]

Hi Pito
Sorry for delay..been busy   .
Yes, i clean up as much as I can. usually my A260/A280 is close to 1.8. As for the adaptors..no they don't say. I think I'll figure it out. I was annoyed this week because my amplified library has tons of primer dimers. Then yesterday, I found the recovery gels from Lonza and that made my day   .
We are using flashgels already so I can't wait to try these guys out. Is anyone using it?

(We are not university and not biotech...we are contractors for the DoD).

[pito]

Ah the PEG trick, yes, you are right there.

DoD?
Is that the department of defense?
Are you doing research on killed soldiers? uh?

[Maddie]

How did you guess?  

[pito]

Maddie, on 07 February 2011 - 03:19 PM, said:

How did you guess?  

I was in the army once...

not american army anyway but for some reason it did ring a bell. No idea why really.
So you are working on soldiers then? or do they research all kind of "corpses"?