Why use 200 units of ligase? - I am going cuckoo lol (Oct/10/2008 )

So I found this paper where they regularly clone shRNA inserts into vectors and since my clone has been a big disastrous failure up till now I decided to give it a go.

So.......I digested my vector like they said, using 5 units of enzyme for 2 micrograms of DNA, then I added CIP the reaction, except that I used a Promega one instead of an NEB one, 4 units directly into digestion mixture with cip buffer 1x.

Then they had double digested annealed oligos to get the inserts with overhangs, since I had my unphosphorylated ones with overhangs because thats how we ordered them, I tried phosphorylating them lol

So some guy on this forum tells me to use 1 microliter of the kinase, buffer and 2 micrograms of insert DNA. I again had a promega kinase which basically told me to dilute it tremendously before using and I didnt know what to do.

Neways then I did a phenol chloroform extraction, got an ok yield I guess

Then I wanted to do the ligationr reaction, they say use 50 ng of vector, 3 ng of insert and behold, 200 units of ligase in 20 microliters volume. So u innocently checked my ligase and it had 3 units/microliter!

I dont understand, some people tell you to not use too much ligase, that 2 units would be enough and others use 200 units lol So i figured I would use as much ligase as I could if I couldnt use 200, so i used 6 units hehehe

So............they transformed using the rubidium chloride method, again.........I had to settle for heatshock, some people say the 42 °C for 90 s is too long so I used 45 s. AS postdoc told me she transforms all of the ligation so I did that, whole 53 ng of it lol


God........this is hilarious..........I am literally going insane with this, im like a molecular moron.


Its kinda fun though, I mean it will be the day I actually succeed in doing this. Its cool to learn about all these possibilities though, inactivate this, dont inactivate that, purify, beware of UV.............



Could someone please explain some essentials so I know what Im doing?? If i think the ligation efficiency is bad doesnt it make sense to use the whole ligation for the transformation? Why would someone use 1 unit and someone else 200? Heatshock in 1,5ml eppi for 50 s better than 90s?

Phosphorylation of oligos? how many units per microgram?

Im starting to feel like I need...........ehh...why yes a supervisor would be nice hehehehe

Yesterday I asked the postdoc that works near me if I could use phenol:chloroform:isoamyl pH 8 for my purifications:

answer: in 20 years of experience Ive never checked the pH.

Asked my boss why someone would order oligos that were longer so that they could double digest them instead of ordering them with overhangs

answer: Oh.I dont know

I need a drink!!!!!


I wish you alla great weekend!!!!!!!!!

All I can say that, I was fine till I started reading your post and now, even I think I should go for a cup of coffee..

Honestly, I still don't understand what you are doing but if its cloning that you are talking about then I could post some of my method but for ligation using 200 units of ligase is total insane. (BTW, where did they find so concentrated ligase stock?) Could you cite that article please?? I use only 1 unit and its too much.
For ligation, use 1:5 and 1:10 vector to insert ratio (I think you have typo as you have other way around)

About your Phenol:Chloroform question, it should be between 6.7 and 7.9. You see a layer on top of your mixture which is buffer and a barrier from the air. The litmus test for this mixture is, if your mixture is pink in colour, throw away, its useless. if its colourless, use it, don't bother about pH and for the sake of your labmates, do NOT shake that bottle. (We had a 'piece' in our lab who would shake the bottle to mix both the layers before using it)

Long Oligos, depending on the need but I think, they just have too much of money or they are plain lazy and the boss doesn't know what they are doing !!

Post back with your specific questions.

1. One Weiss unit of T4 DNA ligase corresponds to 67 cohesive-end units as defined by New England Biolabs. If the paper you are following was using NEB ligase, then 200 Units = 3 Weiss Units (definition that every other ligase supplier uses). It's still overkill. NEB sells ligase at 400 and 2000 Units/uL which = 6 and 30 Weiss Units/uL, respectively.

2. Too much CIAP. According to Promega, "dilute sufficient CIAP for immediate use in CIAP 1X Reaction Buffer to a final
concentration of 0.01u/μl. Each picomole of DNA ends will require 0.01u CIAP.
(1 μg of 1,000 bp DNA = 1.52 pmol DNA = 3.03 pmol of ends.)"
2 μg of 3,000 bp DNA vector = 1 pmol DNA = 2 pmol of ends. Therefore, you need 0.02 Units of Promega CIAP. Note that the required Units varies according to the supplier. 4 Promega Units = 200x too much enzyme.
CIAP and BAP are very difficult to purify and are frequently contaminated with exonucleases that nibble the ends of the DNA when too much enzyme is used, therefore destoying the restriction site.

3. You need to stop thinking about ug and convert everything to number of molecules and number of ends. You want to ligate equal numbers of ends of insert and vector.

Hi!

Lol they wrote down 200 U and I looked at my ligase tube and it said 3u/microliter. Now I know, promega sells in weiss unit..........NEB in cohesive end unit, 1 weiss unit are 67 cohesive end units...........so..........yeah I should have use 1 microliter of my ligase hahahahahaha


Also no it wasnt a typo, my insert is only 60 bp long and my vector is 6000 bg long. When I used a formula to figure out what to use, 1 ng was the result. In that protocol they use 3 ng. A ta in my lab told me to use this much and a PhD told me to bombard the mix with small inserts so to use 2 micrograms.

Not shake the bottle? Lol I got the phenol:chloroform:isoamyl mix from someone and he specifically instructed to shake the bottle before use. Then I asked this girl and she even showed me how to do it, she shook it vigorously for a minute and told me to use the milky mix which is what I did, and I got a 60 % yield of DNA.


Now I REALLY need a drink, it seems that I am going to work my way through all the possible mistakes in cloning.

I know.................I just realized that I need to keep things cool and think things through properly. I think ive been letting my negative perception of the lab get in the way too much of things.
I should just think I do instead of trying out too many different protocols incorrectly.

Thank you a lot for ur input!!!!!!!!!!

@tfitzwater,
Very good answer. I forgot about Weiss units long time ago but thanks for reminding everything.

@nanu nana,
I am still surprised at the phenol:ChCl3 bottle shaking stuff. Ask them following questions,
1. If they have opened fresh bottle of this mix, if yes, did it contain this layer right from the beginning or they added after opening it.
2. What is that layer and what is the function of that layer.
3. If they are aware of solubility of all these ingredients among each other.

Now the answers,
1. When you get the bottle from manufacturer, it doesn't contain this layer, it is shipped separately. You should add it immediately after opening the bottle.
2. That layer is a buffer to prevent air contact with the mixture. If you guys shake the bottle often then your mixture will have very low shelf life (as it will turn red due to oxidation due to frequent contact with air)
3. I don't know this thing either but in my understanding they are perfectly miscible (and so you have one mixture instead of layers)

Hi!


So quite a turbulent morning today,

I asked one of the oldest TAs about the phenol:chloroform thing, and instead of answering my question regarding the shaking she told she wouldnt do it.

Do what ?I ask
Mix phenol:chloroform
Huh?
Chloroform inhibits phenol
Oh but you can buy the stuff mixed together
That is just silly, chloroform inhibits phenol, I wouldnt do that.
Uhm...........ok so the companies that offer this product have made a mistake

I went in search of further guidance, so I asked the guy that bought that mix
Hello Morritz! Is this upper phase that I see the one used for the Ph of the mix?
Yeah why?
Oh because I read on the Sigma website that one is supposed to remove it after youve mixed it once??
(http://sigma-aldrich.custhelp.com/cgi-bin/sigma_aldrich.cfg/php/enduser/std_adp.php?p_faqid=1727&p_created=1210869772&p_sid=Vk8hldgj&p_accessibility=0&p_redirect=&p_lva=&p_sp=cF9zcmNoPSZwX3NvcnRfYnk9JnBfZ3JpZHNvcnQ9JnBfcm93X2NudD04LDgmcF9wcm9kcz0
0Miw0NjgmcF9jYXRzPSZwX3B2PTIuNDY4JnBfY3Y9JnBfc2VhcmNoX3R5cGU9YW5zd2Vycy5zZWFyY2hf
bmwmcF9wYWdlPTE*&p_li=&p_topview=1)
Really?
Thats what it says in questions and answers, so when you shake it your mixing the tris buffer again
Oh well thats how i learned this, you shake, everything turns milky, a slightly more milky phase forms at the top and then you take some from the middle of the solution.
Uhm.................


So technically you mix the tris buffer once, the phenol:chloroform :isoamyl mix turns acidy, and you either remove the upper phase or leave it there so the phenol:chloroform:isoamyl mix doesnt have the much contact with the air. In any case mixing isnt really the way forward from what im understang......

gees monday morning........hehe

The tris is to normally up ur pH so that it's suitable for DNA extraction at least from what i have gotten from Omnipur.

chloroform inhibits phenol?! In what application?
because if it's for DNA extraction , chloroform is a good buddy for phenol. help to increase the density .

u could aliquot that P:C:I in small bottle to avoid frequent airing by the way!

Phenol (C6H6O) has a formula weight of 94.11 and is fairly soluble in water (approximately 1 g/15 mL). Phenol will absorb up to 12% water before forming a two-phase system. This means you will lose up to 12% of your RNA or DNA (not including nucleic acids in the aqueous volume and interface left behind in pipetting off the upper layer) in a plain phenol extraction unless you reextract the phenol phase. Phenol enters the aqueous phase (8-10%) and must be removed by a final chloroform or ether extraction. Note that because phenol is slightly soluble in water, repeated buffer exchanges will reduce the volume of the organic phase.
Both phenol and phenol:chloroform cause proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase. Phenol:chloroform is more hydrophobic than phenol alone and prevents poly(A)+ RNA from partitioning into the interphase due to formation of insoluble RNA-protein complexes and reduces cleavage of the poly(A) tail.

The high hydrophobicity of chloroform also enhances the separation of the organic and aqueous phases and thereby increases recovery of RNA contained in the aqueous phase. This effect can be employed when dealing with thick flocculant interfaces, by adding an extra volume of chloroform to the sample. The interface will pack tighter in a 1:3 phenol:chloroform mixture.

Phase partitioning of nucleic acids is also pH-dependent. At pH 7.0 and higher, RNA and DNA will partition into the aqueous phase, but below pH 7.0, DNA will be denatured and precipitate into the organic phase, leaving 2’-OH RNA in the aqueous phase.

See this excellent explanation: http://bitesizebio.com/2008/02/12/the-basi...traction-works/

I suggest that you add 0.1 g of 8-hydroxyquinoline to 100 mL bottles of phenol, 0.4g of 8-hydroxyquinoline to 400 mL bottles of phenol to reduce rate of oxidation. This changes the color of the phenol:chloroform layer to yellow so you can tell the aqueous layer from the phenol layer and acts as a preservative.

Wooow thank you!
Ok.........
So this is the one we have :http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/P2069

From what ive understood, I use the equilibration buffer that comes with it to adjust the phenols pH to 8, which is the one you should use for DNA extractions (my boss had told me that the lower the pH the higher the DNA yield).

The one I have still containes the equilibration buffer, they just left it there instead of removing it and it is a upper phase that might actually protect my phenol:chloroform:isoamyl mix from oxidizing.

So..........I shouldnt shake the bottle at all...........I should go through the upper phase and pippette from the phenol:CHCl3:isoamyl mix.

Then.........it would be good to perform a chloroform:isoamyl extraction to get rid of possible phenol rests. Unless the Phase lock gel eppis that I have do the job.

For RNA, I will use pH 4.5

hehehehe