Ligation problems using NEB Quick Ligase Kit - (Oct/11/2007 )
I am having some problems ligating a 1020 bp insert into a 6 kb vector. Every time I try the ligation I get little or no colonies using the NEB Quick Ligase kit. I've used insert:vector rations all the way from 3:1 to 10:1. I transform into TOP10 (Invitrogen) E. coli and I know my competent cells are OK because I have done control transformations. I know my ligation is working because I cut my vector with BamHI (cuts once) and then I re-ligate and get a lot more colonies on the reaction that I added the ligase to than the one that I didn't.
My ends are KpnI and SphI. I gel purify both the insert and the vector with a Qiagen kit and elute in EB buffer (10 mM Tris-HCl pH 8.0). The space between the enzymes is 30 bp in the MCS. I know it cuts because I have the insert in one plasmid and I am moving it to another plasmid with a different antibiotic resistence marker. It is because of this that I know my insert is not toxic. I can express it fine in the plasmid with Kanamycin resistence, but I can't ligate it to plasmids that have Spc or Gent markers.
Here is my reation:
1.5 ul insert 5:1 ratio insert:vector
1 ul vector (50 ng)
10 ul 2X Quick ligase Buffer
Mix by pipetting gently
1 ul Quick T4 ligase
Mix again by pipetting gently, spin down briefly, incubate at RT for 5 min. Chill on ice briefly, then transform TOP 10 E. coli with 4 ul of Ligation.
I am using the Quick Ligase kit from NEB and I've also tried T4 Ligase from invitrogen with no luck. This is very frustrating and has put me behind more than a month. Please help me out. I'm pretty desperate. I've also tried different plates.
The first thing I'd do is switch to normal ligase rather than quick ligase. You don't need quick ligase for cohesive end ligations. The normal ligase reaction is cleaner and will work at room temperature in 10-30 minutes.
The second thing I'd do is make sure that my target plasmids were functional with the antibiotic plates I was using. Transform with the uncut target plasmid and make sure the anitbiotics are right.
I'd next make sure that my transformation efficiency was high. How many colonies/ug of plasmid are you obtaining? It should be 10^8 or more.
I'd make sure I was not trashing the vector and insert during gel isolation. In fact, I wouldn't even DO gel isolation. You should be able to clone this insert into a different resistance plasmid without purifying anything. You should be able to mix the target and source plasmids together, cut with KpnI and SphI for 1 hour. You can't heat kill the KpnI, so you'll have to purify the DNA. Add 1 ul of 500 mM EDTA and 1 ul of 20 mg/ml proteinase K to the mix, incubate for 1 hour at 37, 20 minutes at 70, and ethanol precipitate & wash the DNA. Then use 20 ng of the mixture in a 10 ul ligation with normal T4 DNA ligase. Transform with 1 ul of the result and select for colonies with the correct resistance. The vector/insert ratios don't matter very much.
You could phenol/chloroform extract the digestions instead of using proteinase K. Or you could switch to an enzyme which can be heat killed (my preferred choice).
I'd guess the problem is UV trashing of your DNA while cutting bands. Fewer steps means fewer things to go wrong. Less purification is easier and more likely to work. You have strong selection and double cut vector, use it.
Don't I have to worry about the ends from the plasmids ligating together to make one 12 kb plasmid that has both resistence markers? Does this mean I'd just have to screen a lot of colonies?
I know my transformation efficiency is good.
My uncut plasmids both grow fine on their respective plates.
I had a feeling that the UV/EtBr might be the culprit.
Instead of ethanol percipitating the DNA can I use a Qiagen column to purify the DNA after treatment with EDTA/protease?
I checked my plasmid and I can use SacI instead of KpnI and I could heat inacitave at 65. Then I would not have to clean up the reaction, right? I could just go strait to the ligation?
Thank you for your reply. I will let you know how it ends up.
Double length plasmids don't replicate. The two origins are, in combination, lethal, apparently. The major background is likely to be partially cut target plasmid which religates. Colony PCR will let you screen quite a few colonies at low effort, if you have to, but I'd guess you can do only a few and get results.
Yes, you can column cleanup the restriction digest, and perhaps even avoid the proteinase K or phenol treatment. But the heat kill is far easier if you can do it.
I was worried about the plasmids ligating together. Good thing to know that I don't need to be. I can use SacI instead of KpnI, so I can heat inactivate at 65 for 20 minutes and then procceed directly to the ligation. I'll use regular T4 from invitrogen. Thanks again for your help. I will let you know how it turns out. Hopefully, I'll have my plasmid.
IT WORKED!!! Tons of colonies when I transformed my ligation. I screened 10 colonies by restricition digest and 7/10 had the insert I wanted. Thank you so much! I can now continue!!!