Cloning problem, sticky - blunt ends ligation fail . - (Feb/12/2013 )
I've a problem with the ligation of a 650 bp insert in a 12Kb Plasmid . Both of them have been digested with BssHII ( blunting cut) and SwaI ( sticky cut) and then eluited from gel.
So far I've neve obtained any colonies after transformation. The ligase enzyme works perfectly in fact i did the ligation of 1 Kb invitrogen marker for 3 hours at room temperature without any kind of problem. Even all the positive controls of transformation works perfectly. So my doubts are about Ligation reaction.
Now I'm tying to dephosphorilate plasmid and phodphorilate the insert, respectively with a CIP phosphatase and a T4 kinase.
In last ligations the total amount of DNA ( vector : plasmid 3:1) was about 300ng. (maybe too much for the blunt end ligation??) .
I think even that the large size of this plasmid compared to the small insert one could increase the difficulty of this kind of cloning.
Anyone has some idea ???
Thank you for your attention .
Blunt end ligations are quite inefficient, try a couple of days at 4 deg C or up to a maximum of 16 deg C for overnight. 300 ng is a lot for a ligation, the usual recommendation is to keep the plasmid below 50 ng.
Large plasmids can be hard to ligate into as you have relatively few ends per ug.
Make sure that it is molar ratios of plasmid:insert you are working with, not ng ratios.
In our lab we do blunt end cloning but with small plasmid. we also use CIP but instead of T4 kinase we use PNK(poly mucleotide kinase)..But it should work fine with T4 kinase also . We face difficulty when PNK or CIP treatment is not proper, we get lot of re-ligation. I would suggest since its a big plasmid, you can try with different ratios of vector and insert.
thank you both ...
today I'm going to ligate newly, after the use of CIP and
I'm thinking even to do a double-day ligation, the first at room temperature for the sticky ends and the other at 4 degree ON for the blunt ones ... maybe it will be more accurate..
Just wanted to share the following. I had a horrible time trying to blunt ligate a repeat sequence into a vector recently and found the way to get it done when all else failed. I used all sorts of ratios and nothing, all temps and nothing, used NEB blunt/TA ligase and nothing. Finally found the following article and then just adaped it.
Increased cloning efficiency by temperature-cycle
First make sure you are blunting efficiently. I had to blunt my vector and insert. I used NEB quick blunting kit. Make sure that your DNA quality is good after gel fragment isolation before you blunt. Sometimes there are left over salts in gel fragment isolations that can interfere with blunting. Also, save some of your blunted vector prior to SAP so that you can use it as a control. It will tell you if your blunting reaction is working. Ligate some of your non SAP blunted vector to see if the ligase is working and that the blunting reation worked. Nothing fancy needed for that ligation just 50ng vector and some ligase in 10uL reaction with buffer. For the real ligation, use the following:
I use T4 ligase from promega at 3u/uL conc. This is my protocol.
Do 1:3 or 1:6 ratio, I used 1:3 because I didn't have much insert left, but it migh be better with more insert.
50ng vector to whatever amount insert needed.
Used the 10X reaction buffer that came with the ligase. Nothing special.
Add 1.5 units ligase
I added 50ug/mL BSA to the reaction. (just use some of the stuff that comes with your NEB restriction enzymes, diluted of course)
Then I put the reaction in a pcr machine with the following program.
No heated lid, 10C for 30sec, then 30C for 30 sec, cycling for 12-16 hrs.
I didn't heat inactivate the ligase and I used Top 10 chem comp cells at 1uL ligation mix per 50uL cells. Might work better with 2uL.
Hope this helps someone! Pass it on!
Sorry forgot to add that I add .5uL ligase to my 10uL reaction. This is to crowd the pieces together and increase the likelihood of getting what you want.