Trouble with ligation - (Dec/15/2006 )

now its 5 months..................i am trying to ligate 2.3 kb fragment in 6.5 kb vector. its very simple ...........but i failed all the time .

my procedure was

Vector digestion :
vector (6.5 kb) 1.2 µl (5 ug) (2.33 pmol)
NcoI 1 µl (10 unit)
NcoI buffer 10 µl
MilliQ 87.8 µl
total 100 µl
incubate 37ºc, 1 hour

check

Phenol:chloroform treatment

Ethanol precipitation

Dephosphorylation:

DNA ppt
CIAP 10 µl (10 unit)
CIAP buffer 10 µl
MilliQ 80 µl
total 100 µl

incubate 37ºc, 15 min

incubate 55ºc, 45 min

add 5 µl SDS (from 10% SDS)
add 1 µl EDTA (from 0.5 M EDTA)
mix
add 100 mg Proteinase K
incubate 37ºc, 1 hour
Phenol:chloroform treatment (2 times)

Ethanol precipitation

Blunting
DNA 8 µl
Buffer 1 µl

incubate 70ºc, 5 min
incubate 37ºc, 5 min

add 1 µl T4 DNA polymerase
incubate 37ºc, 5 min (exactly)
vigorously vortex

prepare for ligation



Insert preparation:
insert (2.3 kb) 8 µl (5 ug) (6.5 pmol)
Buffer 1 µl

incubate 70ºc, 5 min
incubate 37ºc, 5 min

add 1 µl T4 DNA polymerase
incubate 37ºc, 5 min (exactly)
vigorously vortex

prepare for ligation


vector: insert= 1:10

I DONNO WHERE IS MY MISTAKE

Hello, sometimes ligation can be really hard. But we don't konw why!!!!

After my restriction, I purified my DNA by agarose gel purification and check the result on gel.
Always inactivate your enzyme!! When you dephosphorylate your plasmid DNA, you need to test if it's ok or not. Just realise a ligation with plasmid alone. You must not have colonies, positif control of your dephosphorylation.
If it's ok and if you haven't colonies for the ligation with the insert, you have a problem for your blunting. Realise also a transformation with an other plasmid who works easealy like pBluescript, positif control of your transformation. Normally T4 DNA polymerase is the good one but you can also use Kleenow.

Normally the first I never use control for the different step but if it's don't work well. I realise a positive control for each step just to know where is exactly the problem.

Hope to help you

biofred

Are you sure you vector has a concentration of 4.1 ug per ul? That is realy high. How did you get this concentration estimate? If by spectrophotometer, I would suggest your try running said DNA on a gel. If by gel, I would suggest going by spectrophotometer.

And if these value are real, would you mind sharing your growing conditions... please? That is a rather high yield.

I assume that your vector has been completely digested? 1hr, for 5ug DNA even with 10U enzyme is rather fast. My calculations throw a value of 2hr for complete digestion, no faster... but if all the DNA cuts all the DNA cuts...

Then the phosphorylation NEB's rule of the thumb =
0.1U dephos 1pmol DNA in 50ul within 60mins at 37 Celsius

I have recalculated the number of pmol of 5ug DNA with 6500bp lenght... the value I get is 1.27pmol rather then 2pmol. The calculations were done by a macro I wrote and use at work. I believe it is accurate. Please recalculate, an error has occured.

But assumming your value of 2pmol is the correct value, then according to NEB's rule, the DNA should only be dephos for 1.2mins rather then 15min. Thus according to my calculations the DNA is way over dephosphorylated and thus the DNA overhangs are probably badly degraded.

I would dephos all 2pmol of DNA with 2U CIAP for 12min in a volume of 50ul.


The next thing I dissagree with is the use of EDTA to deactivate the CIP. CIP has been stopped by EDTA, degraded by protenase, and denatured by phenol/chloroform (twice!) This is far too much.

Don't use EDTA inactivation (ever!) in DNA cloning. Any residue EDTA will inactivate the ligase enzyme. So don't use it. You do not need to conduct the protenase step (which should have been conducted at 55 Celsius). And you only need to do the phenol/chloroform step once. Each phenol step results inevitable lost of some faction of DNA.


Blunting by T4 DNA polymerase full fill in?

Does your buffer have dNTP added? The NEB T4 DNA polymerase buffer does not have dNTP, you have to add this on your own. Also this enzyme is suppose to work at 12 Celsius... Elevated temperatures (37 Celsius) will lead to recessed end by 3'-5' nuclease activity.I feel that the volume is too small for the amount of enzyme added. The glycerol concentration of 5% (from the enzyme) may be inhibitive. 5 mins is also short.

Any reason not to inactivate this enzyme? I have never tried leaving this particular one active in my ligation mix. But I don't like the feeling of something that could make 3' overhangs on DNA following prolong exposure.


What are you ligation conditions? How much DNA, how long and at what temperature?


I calculate insert 2.3kb insert of 5ug at 3.58pmol rather then 6.5pmol.

Other points to add,

did you gel purify your DNA? There are alway some protein, RNA and DNA junk and denatured plasmid DNA that needs removing.

[Hi
many thanks for your reply.
i am answering u step by step
yes i am sure about the concentraiton of my DNA sample. I mesured it by spectrophotometer. I used 50 ml YT broth with appropriate antibiotic and culture for 18 hours with vigorous shaking at 37c. I used alkaline lysis method and cesium chloride gradient and centrifugation for 18 hours at 45000 rpm to prepare the DNA sample. even sometimes i got 8 ug/ul DNA.

yea i wrote that the vector digestion time was 1 hour........but its a ideal time, usually i digest up to 2 to 3 hours. and i checked it by gel electrophoresis and then i purified the digested vector.

for dephosphorylation i followed Sambrook...............so i just did what sambrook says. in our lab me and everybody are doing this in the same way.........i also did it many times but only in this case i failed again and again.

i calculated the pmol from calculator of promega
http://www.promega.com/biomath/calc05.htm

in sambrook they strictly said to follow phoh:chcl3 step twice after dephophorylation.
according to sambrrok proteinase step is either 55c for 30 min or 37c for 1 hour.

we use DNA blunting kit from TAKARA.

my ligation condition was vector: insert= 1:10, 16c for atleast 1 hour upto overnight.

i use Takara mighty Mix for ligation.

i purify inset from gel, i was careful to purify the DNA form gel.

thanks you a lot
i would be grateful if you can design a procedure for me
now i just feel helpless

thanks for the growth conditions. Just one question on that matter, what is your total DNA yield? How many ul of 4ug/ul DNA do you usually have?

I am happy to lend you a hand with your cloning, however there are a few things I need to say...
1- As you know, blunt end ligation is not easy. Is there any way possible to go by a conventional sticky end ligation?

Would you mind buying primers to amplify said insert via PCR to give it NcoI sites? Is that even possible? (ie does the insert have NcoI sites) It would certainly help if I had the sequence and an annotated map of the plasmids. The new plasmid you are making, is it for expression? thus requiring maintainance of frame order.

2- the insert. Could you tell me more about it? Is it an insert cut out of another vector or is it a PCR insert? What kind of ends does it have?

I found out why there was a discrepency between the two calculation. I am calculating mols of DNA, while the site actually calculates mols of DNA ends.

thanks again. my total DNA yield was about 800 ug in this case. after CsCl gradient i always precipitate DNA in 200ul TE.
answer of ur ques.

1. there is no way to perform sticky end ligation.
, so it has a particular frame order. actually NcoI site was constructed by PCR to maintain the start codon.

2. about insert: insert was cut from another vector and SphI site was constructed by PCR for start codon beacuse it has already a NcoI site . so blunting is the only way.

i am really hopeless now.........

Due to the difficulty of the current cloning strategy, (blunt end cloning especially one where your vector is rather large, full fill in of vector and 3'nuclease of insert), I think it would be prudent to investigate (again) the possibility of any other ligation strategy. Could I have a look at an anotated sequence of your vector and insert-plasmid? Genebank format would do.

It would really help if I had a map. Are you fusing two proteins together? Are you fusing a vector ORF (maybe adding a Tag of somekind) with your INsert ORF. Because you stated that the NcoI site was constructed into the vector.. indicating the the NcoI is none native.... and thus can be changed.

If your are just introducing a new ORF into a blank expression vector (not making a fusion protein), there is no need to maintain the NcoI site.

Are there sites upstream to the NcoI site that you could use?

It it at all possible to PCR your insert to include of BspHI sites? Does your insert have BspHI sites? BspHI produces overhangs that are compatible with NcoI.

I try to avoid as many purification steps as possible.
my suggestion: restriction, heat inactivation, just add klenow and dntps to the restriction, heat inactivation, just add phosphorylase, heat inactivate if using a heatlabile phosphorylase (really good this time, i usually do it 1hr @70degC). if not, purify here. ligate. i use usually 20myl rxns, always works great!
greetz