For the last 2 weeks I have been trying to clone a PCR product into a vector digested with blunt PmeI. I digest the vector for about 6-8 hours, then add CIP to prevent religation. Next, vector is purified with a kit and DNA concentration measured. The PCR products are prepared using Phusion Polymerase, purified using kit, phosphorylated with T4PNK for about 1-2 hours and then inactivated at 65C for another 1-2 hours. I do not purify the PCR after reaction with T4PNK, and just use it after inactivation for about 5-8 hours ligation at RT.
The vector to insert molar ratio is 1:10 for 10ul ligation reaction. I transform with the whole 10ul volume. I tried to transform DH5Alpha chem. competent, TOP10 electro and chem competent, XL10Gold chem. competent, and the result was the same, no colonies on insert ligation plates. I want to add that I'm cloning 5 different inserts at once and every time prepare controls which is: + (vector, buffer, ligase, and water), -(vector, buffer, and water).
Interestingly, I'm getting colonies on my control(+/-) plates with greater number on (-) control! Also, I've changed ligase and ligase buffer, which had no effect. I did blunt end cloning before with the same methodology and never had this much of trouble. Any suggestions?
Hi
What's the size of the PCR product ? Depending on which vector you use, if it's too big you may have problems.
Hi Alexis,
The vector is 6.9kb and it is pcDNA3.1 that I modified for BAC recombination experiments later down the road. My inserts are 1.1kb and 1.5kb I know that the size of the vector may be a problem but it is not that big either. Before I've been cloning larger inserts in to even bigger vectors. Again, your point is valid but to not have any colonies after ligation...? I can not figure out whats going on.
-I don't know for this Phusion Pol, but many polymerases add As in 3', and then your insert is not blunt. Some vectors are sold linearised with "cohesive" (not much) Ts.
-I always do blunt ligations O/N @ 16°C, it's supposed to work better.
-Did you try not using CIP? In my experience, this dephosphorylation step never helped, and often actually prevented ligation, probably because of a lack of purity of the phosphatase (contaminating nucleases etc).
-If your enzyme is good enough, overdigestion can be a problem. 6-8 hours seems a lot, but apparently you have an insert problem, not a vector problem (control plates have colonies).
-Don't worry, it always gets in, in the end.
I agree with avoiding using CIP. If it's not a product size or amplification (Phusion pol is perfect for blunt end inserts) problem then this ligation inhibitor might be the cause if the purification step is not sufficient.
I have also used a blunt kit for 2.5 to 3.3 Kb cloning by amplification with Phusion (with blunt end).
- As vincent I did a ligation O/N at 16°C
- Never used a CIP
- What kind of vector you have (linear or circular)? If linear, I think to don't have to digest it.
- Is it a blunt from Invitrogen?
- You can also decrease the amount of your insert.
I agree with the above suggestions. I don’t see why you are obtaining more colonies in the controls than in your test sample. If I were you, I would still perform some colony PCRs only to validate statistics.
It looks like your restriction digestion is not complete. You have a lot of background from your control without the ligase. Linear plasmid has very low transformation efficiency to explain this result. You have to make sure that the RE is working properly and also the T4 PNK.
I have read in your previous response that you have made a recombinogenIc pcDNA3.1 plasmid for downstream applications. Is it possible to introduce the recombination sites into the primers and make the recombination in vitro? I mean the way gateway technology works. I also suggest you explore LIC (ligation independent cloning) using Nicking enzymes.
Dear All,
Thank you for all the suggestions, I really appreciate that! To answer some of the questions;
Vincent, Alexis and Justin-I've never tried blunt cloning without CIP. I'm just afraid that I will have a carpet of colonies that will will not contain any insert...
Abdelhalim- Unfortunately, I do not have any comonies on ligation plates otherwise I would be analyzing everything that would be growing there... For recombination; in our lab we were using GalK system that to knock out genes in BAC containing cytomegalovirus sequence. The next step was to knock in a mutated version of earlier KO gene. Unfortunately, the system is malfunctioning so I designed a system where I KO genes with Ampicilin cassette and in the second recombination insert my gene of interest with Kanamycin/LacZ cassette. The KanLacZ cassette is in the midified pcDNA3.1 plasmid flanked on both sides with FRT for further removal of KanLacZ from the BAC sequence after previous selection. So to to do create the knock-in fragment I first need to clone it in to the modified plasmid. Thats where my cloning problems start...
Hi Robert.
I have done a lot of successful blunt end ligations and agree with what others have suggested. ON-Ligation using NEB T4-DNA ligase kit I do at 4°C. I also get results with 1h at RT. But, I'd always recommend CIP the cut vector, even with sticky ends. I find it increases the ligation efficiency.
Here's what I think might solve your issue: Try adding a blunt end site to your PCR primers plus 1 or 2 additional nucleotides to increase cutting efficiency (of course use the same site and this site should not cut in the PCR product). That way you avoid the phosphorylation reaction. I once even order phosphorylated primers for a lot of money and tried to blunt-end clone the deriving PCR-products - it never worked. Since then I prefer the RE-digest...
Robert,
If you only need a cassette then I strongly recommend to you to try overlapping PCR which generate your exact product in two PCR steps. You don’t need to add anything only if you want to. I was producing cassettes by assembling up to 5 different PCR fragments and up to 5 Kb in lengths to be integrated into the genome in less than a one day. You can amplify all fragments to be assembled separately and adding homology sequences to the primers, gel purify and PCR with oligo from the first and last fragment. Your cassette is ready. I have never tried to mix more because I didn’t need to. If your cassette is bigger, then you can try Gilson assembly but I think people are having problems with it. So, if you need more detail on overlapping PCR I can tell you more
You can also try using different molar ratios: 1:1, 3:1, 6:1 (insert:vector)...
I've the same problem, but, after days and days of troubleshooting, I definitely changed protocol: goodbye restriction enzymes! I switched to Exponential Mega Priming cloning.
For more info, look at this ;)
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0053360
P.s.: it seems you have pDNA not completely digested in your controls
Hi Robert,
I tend to agree with Christopher, if you don't need the insert in frame to express it, then I've always had the best luck engineering a restriction site onto my oligos for PCR. That way you can bypass the phosphorylation step all together...simply amplify, digest, and gel purify your fragment. I've found it to be the cheapest insurance to successful cloning projects. The Phusion enzyme says it results in blunt ended pcr products...but you never know. Alternatively, when I have issues with cloning, i always do what Nicola has suggested and alter the insert:vector molar ratios and that usually solves the problem. Sure, it's an additional amount of work, but one of the ratios usually works. It's not pretty or elegant, but problem solved.
Good Luck!
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