Ligation SalI / XhoI - (Jul/18/2008 )

Hi everybody !

I have a problem with cloning. I am trying to insert a Neo cassette of 2kb into a plasmid of 5.8kb. The plasmid has been digested SalI and dephosphorylated using CIP. The plasmid carrying the Neo cassette has been digested using XhoI to take the Neo cassette, and XhoI site is compatible with SalI. Both plasmid and insert have been purified on gel. I did my ligations in a volume of 10 ul and I have tested two ratio of insert/plasmid : 3/1 and 5/1.
Unfortunately, I have obtained any colony !
I know that my transformation worked (since I got colonies using a pCDNA vector).
I know that dephosphorylation and ligation worked (since unphosphorylated vector gives any colony and that phosphorylated vector gives many colonies...).
Have you some ideas to explain my results ???
Thanks a lot for your answer.

Christophe.

Christophe,
You have a tough case :-)
I would start solving it by running 5 ul of gel-purified insert and vector on a gel to check that you did not loose your DNA.
Andriy

First, what are your ligation conditions? How long? What temp? How much DNA are you putting in? What ligase? I prefer to use the T4 (not quick) for 4 hours on my bench. I only put in a maximum of 50ng template and the appropriate amount of insert. How long are you growing your transformations? You may want to grow them for longer than the one hour. What is your antibiotic selection? If kan, put your plates back in the incubator for another night (no colonies=nothing to lose). I had a labmate that was trying to do a ligation for months and had no colonies until he forgot the plates for an extra day. Turns out that the plasmid makes bacteria grow very slowly (hard to amplify as well) but he did get plasmid with the ligation. The only other thing I can think to ask is are you calculating the molar ratio of your plasmid to insert? May seem stupid but I've seen people doing a concentration ratio, not molar which is very different!

Try growing the transformation for the full hour, especially now that you are having trouble. It certainly isn't going to hurt. I'm not sure how much DNA you are putting into the ligation but too much can inhibit the reaction. This is a tough one because you know your digestions are good (insert is not pcr product). Your purified and I assume properly quantified from a gel and OD260 and seem to be doing everything correctly. The only other thing I can think of is that your insert is taking on a secondary structure which is inhibiting the ligation. Try heating up the purified insert, set up the ligation and quickly add the heated insert. It's a long shot but may be worth a try if you've done everything else. Another stupid one but are you sure the ligase is still good? Has the buffer been freeze/thawed multiple times? The buffer has ATP which in necessary and degrades with each freeze/thaw. I aliquot mine out. I know this is reaching at straws but you never know what little detail is potentially the problem, especially if you happen to be in a lab where these enzymes and buffers are common items. You never know if someone accidentally left the buffer or enzyme out at room temp too long.

Beware the cursed of SalI !

Yes, for using SalI in a ligation, you have now become cursed! This ligation will never work, until you have repented and offered a scarifice of blood, sweat, tears and a chocolate bar... The gods perfer KitKat.

There have been numerous problems using SalI as part of a ligation strategy. Is it at all possible to use some other pair of restriction sites?

It is also possible to conduct a partial fill in and make compatible overhangs

BamHI/BglII/BclI compatability group, once partial filled in (2bp) form compatible ends with XhoI/SalI group.

Note BclI is dam sensitive. So it is usefully only when Dam minus cells are used or with PCR amplified DNA.

NEB website entry for Sal I says it doesn't like PCR products... Check this out, from the FAQ link of the Sal I entry:-

Q1: Is SalI affected by methylation?
A1: SalI is blocked by CpG methylation.

Q2: Is SalI sensitive to pH?
A2: SalI activity decreases if the reaction buffer pH is not between 7.8 and 8.0.

Q3: Is SalI affected by star activity?
A3: Yes, high glycerol, high enzyme concentration, or long reaction times can cause star activity of SalI.

Q4: Are extended digestions with SalI recommended?
A4: Greater than 4 hour digestions are not recommended.

Q5: Is SalI inhibited by nucleotides?
A5: Yes.

Q6: Does SalI exhibit reduced activity on supercoiled DNA?
A6: Supercoiled plasmids may require up to 10-fold more SalI for complete digestion than on linear DNA.

Q8: Does SalI have trouble cleaving PCR products?
A8: SalI has trouble cleaving PCR products.

So, don't have methylated DNA, check the pH, remove any nucleotides, don't use supercoiled DNA, and don't use a PCR product. That should leave you with lots of choices...not. How about another enzyme??

I agree, I have had a lot of problems with SalI too. Just use another enzyme.