LIGATION
http://www.labtimes.org/labtimes/trick/tricks/2013_07.lasso
[rel://files/experiments/ligation.ods] [rel://files/LigaFast Rapid DNA Ligation System Protocol.ashx] [rel://files/PCR Cloning Kit (blunt end)%0A1939645a.pdf] Standard Ligation: Prepare a ligation mix: vector+buffer mix 5x ligase buffer(x) 6 H2O 6 Digested vector 1 total 13
Divide vector+buffer mix into two Eppendorf tubes +/-: + - Vector+buffer 5 5 µL insert 5 0 µL water 0 5 µL T4 DNA ligase (400 u/ml) 1 1 µL Total 11 11 µL
incubate for O/N at 8-16C in the cold room Proceed with the transformation of the appropriate E. coli strain with 10 microL of the ligation mix.
(x) Homemade 5x ligase buffer 250 mM TrisHCl, pH 7.6 50 mM MgCl2 5 mM ATP 5 mM DTT 25% (w/v) PEG 8000 vortex vigorously at RT after thawing
PEG 8000 at 5%(w/v) in the ligase buffer has been shown to improve the ligation efficiency of blunt end DNAs. The reference is a 1986 BRL Focus "optimizing DNA ligations for transformation" Vol. 8 #1 (winter issue). For the ones who cannot get this issue , the buffer was the following:
5x ligase buffer: final 250mM Tris-Cl pH 7.6 50mM 50mM MgCl2 10mM 25% (w/v) PEG8000 5% 5mMATP 1mM 5mM DTT. 1mM
Aliquot and freeze at -20. The conditions recommended for ligations of blunt end insert into vector DNA were the following:
1x buffer 1 unit or BRL T4 ligase ( BMB will do the same job) vector/insert molar ratio =3 ( up to you to change it ) 4 hours at room temp (23-26C) Dilute 3 to 5 fold before adding DNA to competent cells.
++Fermentas http://www.fermentas.com/techinfo/modifyingenzymes/protocols/p_dnainslig.htm DNA Insert Ligation into Vector DNA (with T4 DNA Ligase)
In a microcentrifuge tube prepare the following reaction mixture:
50-400 ng Linear vector DNA Insert DNA (use a 1:1 up to a 3:1 molar ratio of insert DNA termini to vector DNA) 2 µl 10X ligation buffer for T4 DNA Ligase 2 µl 50% PEG 4000 solution (for blunt ends only) to 20µl Water, nuclease-free 0.4-1uL T4 DNA Ligase 0.2-0.4 µl (1-2 u) for sticky ends / 1 µl (5 u) for blunt ends
Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. Incubate the mixture for 1 hour at 22°C (on the bench). Use the mixture for transformation.
Note:
- If the yield of ligation product is insufficient, prolong the reaction time (overnight).
- DNA can be dissolved in nuclease-free water or TE buffer: 10 mM Tris-HCl, 1 mM EDTA (pH 7.8).
- An excess of ligation mixture with respect to competent cells may decrease the transformation efficiency.
http://www.benchfly.com/video/61/make-your-own-rapid-ligation-kit/
Ligation buffers are usually mixed from five simple ingredients. Only one, however, has a major impact on the speed of the ligation reaction: Polyethylene glycol.
If you do a lot of cloning in your lab you may at times wonder how to make your ligation reaction faster, more reliable and at the same time much more affordable. Even if you haven’t, keep on reading – it’s never a bad idea to save time and money. I, for one, can think of a million things to do if I had copious amounts of both these (unfortunately rather rare) commodities. Going to the beach and drinking margaritas is usually at the top of the list. So, how can you save both time and money to make that happen? The answer is so simple, it hurts to look at it: go fast and go cheap!
There are a lot of expensive ligation kits out there. The so-called “rapid ligation kits” are popular because of the added value that the ligation reaction needs neither a controlled temperature, nor a prolonged incubation time. Conventional ligation requires incubation over night at 16 °C. With a rapid ligation kit you can get away with as little as ten minutes at room temperature – dang!
True, these will save you time – but they won’t save you any money. Which means you can go to the beach but you won’t be able to afford the margaritas! So, you might as well stay in the lab and continue with your experiments.
Viscous buffer The reason why the rapid kits work is not because of some fancy Speedy-Gonzales-ligase that is capable of doing what the regular ligase can’t do. Turns out it is exactly the same enzyme, i.e., good old T4 DNA ligase. Rather, the secret is in the buffer: because it is very viscous it slows down the diffusion of the reaction partners, effectively mimicking the effects of reduced temperature. This increases the chances of loose DNA ends matching up, sticking together via their overhanging unpaired nucleotides and getting ligated. Because the sample is not chilled the actual enzymatic reaction is not slowed down; which is why ten minutes are indeed plenty of time for the ligation to occur.
I have applied this protocol literally on hundreds of cloning experiments and it didn’t fail me once. All we need to know, you might say, is the composition of the buffer. Manufacturers of rapid ligation kits will rather cut off their right arm than tell you what’s in it. Luckily, my sense of self-destruction has not quite developed this far – which is almost surprising given that I have spent the last fifteen years in research.
Mixture of lower mass PEGs The key ingredient that will make all the magic happen is polyethylene glycol (PEG). PEG is an inexpensive polymer that is sold as a dry powder. Depending on the average molecular mass, you can get different PEGs, ranging from 3,350 g/mol to well above 20,000 g/mol. Usually, the lower mass PEGs do well in this buffer.
Here is the recipe:
132 mM Tris, pH 7.6 with HCl 20 mM MgCl2 2 mM DTT 2 mM ATP 15% PEG (3350, 6000, or 8000)
This yields a two-fold concentrated buffer. Since the buffer contains ATP and DTT it’s a good idea to aliquot and store it at -20 °C and avoid freeze-thaw cycles. For an average ligation reaction I usually aim for a total volume of 20 μl, ligating 100 ng of purified vector backbone to three times as many insert molecules. Hence, a typical reaction would look like this:
100 ng purified vector 3x molar ratio of purified insert Add ddH2O to 9 μl 10 μl of 2x homemade ligation buffer 0.8 μl of T4 DNA ligase
Vortex briefly, collect by centrifugation and incubate at room temperature for ten minutes. That’s it! Now you can chill the reaction on ice, add your competent cells and proceed with the transformation. This also means that you have just saved a lot of time and money.
Therefore, pack your beach towel, grab your swimming trunks and off you go to “Margaritaville”. Enjoy!
1 - Test ligase activity by ligating plasmid digeste with sticky-end producing restriction enzyme.
In our case, we used plasmids "insert plasmids' names" that were digested by HindIII (Gel 1). This method was inspired by this thermofisher protocol. In order to run the ligation product we used loading dye with SDS (final SDS concentration 0.2%).
Gel 1 - From left to Right: EcoRI digested plasmid - non-digested plasmid - HindIII digested plasmid Sk1B (pos µ1117) - molecular weight ruler - EcoRI digested plasmid ligated with homemade buffer - non-digested plasmid ligated with homemade buffer - HindIII digested plasmid Sk1B (pos µ1117) ligated with homemade buffer - EcoRI digested plasmid ligated with TheromoFisher provided buffer - non-digested plasmid ligated with TheromoFisher provided buffer - HindIII digested plasmid Sk1B (pos µ1117) ligated with TheromoFisher provided buffer
Ligase activity of T4 ligase at 1U/uL seemed to be enough to re-ligate a sticky end plasmid by using either home made or commercial buffer.
2 - Test ligase activity by ligating insert with blunt-end digested plasmid.
In our case, we used a GFP expression cassete amplified by PCR from P2 plasmid by primers 407 and 509 as insert. We used pYPKa digested with AjiI as the blunt-end digested plasmid.
The product was amplified by either supreme NZYTech polymerase or a commercial Taq polymerase (Gel 2).
- The supreme NZYTech polymerase PCR product was ligated to linear pYPKa by using 1 uL T4 ligase at 5U/uL with commercial Thermofisher buffer (Final ligation volume 20uL).
- The commercial Taq polymerase PCR product was ligated to linear pYPKa by using 1 uL T4 ligase at 1U/uL with commercial Thermofisher buffer (Final ligation volume 20uL). The PCR buffer was also different for each polymerisation reaction. Supreme NZYTech polymerase used the manufacturer provided buffer, while commercial Taq polymerase used and home made taq buffer.
Gel 2 - From left to Right: Supreme PCR product - molecular weight ruler - Taq PCR product
The supreme NZYTech polymerase PCR product ligated with T4 ligase at 5U/uL proved to be the successful approach (Gel 3). Apart from the already stated differences, the insert seemed to have been used in greater amounts.
Gel 3 - From left to Right: pYPKa digested with AjiI and Supreme PCR product - pYPKa digested with AjiI ligated with Supreme PCR product - molecular weight ruler - pYPKa digested with AjiI and Taq PCR product - pYPKa digested with AjiI ligated with Taq PCR product - intact pYPKa plasmid
The same total quantity of DNA of each type was used for each well.
After transformation in E.coli competent cell, we were able to confirm the presence of ligated plasmids by colony PCR using primers 577 and 578 (Gels 4 and 5)
Gel 4 - From left to Right: colonies 1 to 12 - molecular weight ruler - colonies 13 to 24
Gel 5 - From left to Right: colonies 25 to 36 - molecular weight ruler - colonies 37 to 46 - positive control (pYPKa as template) - negative control (ater as template)