ligation never works even for single cut vector!Why so!Help! - (Sep/08/2006 )

Hi,

I have a big problem with my construction of new plasmid with my cDNA inserts. The vector is about 4.7kb, inserts about 1.5k which I think is fine. And I design primers with extra restriction sites that match the enzymes in MCS region in the vector. I digest the vector with XhoI and NheI, then purify with gel extraction. At the same time I PCR the inserts with the primers I design, then purify with PCR cleanup kit from Qiagen. For all these fragments and linerized plasmid I run gel and size and purity perfect. Then I ligate ,transform, not a single c,one grow!!! But the transform of the plasmid itself and pUC19 grow very well.

Then I try the control which cut the vector only single enzyme, then do all the ourification again, run gel and determine concntration, then I try to ligate this single cut vector, transform, Still NOt a SINGLE clone grow!! And I assure everytime I do pUC control for x-form. ANd also I tried 3 different ligase, all brand new!! Nothing works.

So i think maybe it's the plasmid it self. I then tried a coworker's totally diffetent vector, the same thing happens!All good until ligation!!

I've been working on this forever!! don't know what's going on here!

So sincerely asking for help!!

Thank you very much!

-plasmid-

I suspect u might b overdigesting ur plasmid as u wrote that even the single digested plasmid, didnt give any clones.

reduce ur digestion time.

U also wrote that u tried a co-worker's plasmid, did u try the same enzyme for digestion or different?

Also can u describe how do u prepare ur ligation mixture.

-scolix-

Hummmmm,, I used to have a similar problem. How big is your ligation reaction?? the glycerol concentration from the enzyme may affect the ligation efficiency. What is your insert to vector ratio?
Some tips that helped me:
I had to do sequential digestions since both of my enzymes were from different companies.
If its the ligation reaction:
Use this formula to calculate the amount of insert and vector in 50uL ligation reaction:
ng insert= (ng vector *kb insert * ratio)/kb vector ----> try not using more than 50ng of vector
I use Taq: Pfu ligases at 1:10 ratio
Ligate overnight at 15 degrees

From what I see, is either your ligation or your digestion, how much are you digesting? you may be trying to digest to much (I did that mistake before, and my single cut did not transformed)

Hopefully this helps somehow

QUOTE (plasmid @ Sep 8 2006, 02:02 PM)

-medchemgirl-

Several things come to mind.....

Firstly
Do you dephosphorylate your vector? If you over dephophorylate, you will trash your ends. Something with the Ligase. Too much is a very bad thing. Less is more.

2
Is your insert PE wash buffer/ QG buffer free? Even slight contaimination from either solutions will will kill all ligation activity. Ligase is a very picky enzyme. I leave the PE solution sitting in the column for 2min as oppose to spinning immediately. I also dry the column form the PE solution by spinning for 2mins rather then 1min. Be careful not to get any wash buffer on the column
I am particularly interested in this point as your single cut control vector failed to work.

3
NheI and XhoI are good enzymes, both digest in NEB buffer2 + BSA. If your DNA is clean, over night digest is fine. These enzymes will not themselves trash your ends. However if you DNA is too concentrated, no restriction enzyme will cut it.

And speaking of ends, is there enough bp around the restriction site to allow the enzymes to cut? This goes for both insert and especially the vector. How close are the two cut sites on the vector?

-perneseblue-

an often-overlooked problem...ligase buffer contains ATP, which can 'go bad' over time. have you tried fresh ligase buffer?

-aimikins-

Thanks very much!

1. I set up my ligation reaction as below:

for quick ligase from NEB: 50ng Single cut linearized vector(around 2ul) and adjust to 10ul by adding 8ul water, then 10ul buffer, 1ul ligase(totally follow the instruction mannual). RT, 5mins

for regular ligase from NEB: 20ng single cut linearized vector, 2ul buffer, 1ul ligase, water add total volume to 20ul. 16 degree 16 hours. I also tried RT 16 hrs.

For regular ligase from Promega, same as above.

2. Also, I thawed buffer on ice, and even has aliqoute right after i thaw them for the first time.

3. the ratio I ever used to ligate vector and insret I tried: 1:1,1:3,1:4,1:8. None grow!

4. when gel extraction, i do let the elution buffer stand 1-2mins, and centrifuge down for 1min.

5. I really don't think I over digest because I tried 1hr, 2hr,3hr,4hr digestion and run the gel. All look good on gel at all, even the longest digestion time give no other band on gel. So I tried 1hr since as short time digest as possible for ligation.

I don't know hoe these info helps, but I'm seriously n depair!

Still thanks for everyone!

-plasmid-

Just out of interest have you checked your E.coli is ok by just transforming good DNA? And also your antibiotic and its concentration?

-Keeva-

Sorry if this sounds like being a smart-ass, but you didn't mention that you actually digested your PCR fragments as well, so if you simply add your sites by PCR, they won't be sticky and thus won't ligate into your sticky-ended vector.

If you did treat your fragment with restriction enzymes but didn't add some 2 or 3 extra nucleotides to the very end, the enzymes might not cut - the NEB catalogue has a list that details wich enzymes need an overhang to cut properly.

DJG

-DJG-