How to increase the efficacy of ligation when using single RE digestion - (Sep/26/2005 )
Hi everyone,
I am trying to ligate an insert that has Xho I - Sal I ends into a vector cut with Xho I. I dephosphorylated the vector (CIP) using a bunch of time-points but the ligation did not work. Also, my insert is as big as the vector, and my biggest fear is that the insert may self-ligate. Do you know what else might be an obsticle?
Thanks in advance!!!
Hi, you cannot dephosphorylate both the vector and insert if you do so, you will never get ligation b/c you need at least one phosphate to do the ligation.
Yes your insert will religate to itself too, but you should not worry about that b/c it will not show up in screening (no antibiotic resistance) you should dephosphorylate the vector as you have done, and make sure the cip is inactivated either phenol xtract/ppt or heat inactivate (CIP may not heat inactiviate well according to other posts BAP OR SAP are good heat-labile alternatives)
Increase the amount of insert in the reaction so that there are enough inserts that self-interaction will not prevent the ligation to your vector... also increase the concentration in your ligation will help improve the chances that the insert and vector come together with ligase more often....
Yes your insert will religate to itself too, but you should not worry about that b/c it will not show up in screening (no antibiotic resistance) you should dephosphorylate the vector as you have done, and make sure the cip is inactivated either phenol xtract/ppt or heat inactivate (CIP may not heat inactiviate well according to other posts BAP OR SAP are good heat-labile alternatives)
Increase the amount of insert in the reaction so that there are enough inserts that self-interaction will not prevent the ligation to your vector... also increase the concentration in your ligation will help improve the chances that the insert and vector come together with ligase more often....
Thanks for your answer (and for citations also.....). Interstingly, not everyone realizes that some people may just START digging into big science. But thanks, anyway......
When you say the ligation "did not work", what do you mean by this? Did you get zero colonies when plating the transformation mixture?
What vector are you using?
How did you prepare your insert? Is it a PCR product?
What vector are you using?
How did you prepare your insert? Is it a PCR product?
Hi HomeBrew,
The number of colonies on the empty vector plate was not different from my test plate. I thought that the CIP timing could have been an issue, but after having increased the time of dephosphorylation up to 90 minutes, I got the same result. The vector seems to keep on self-ligating..... CIP is new, so it should be the problem....
I got the vector from UCLA. It's called pBabe... My insert is basically a construct that I engineered in vector pWZL. I cut the insert out of pWZL vector using XhoI - Sal I double digest that unfortunately generates compatible ends. The insert is about 5 KB (so is the vector) and looks like it tends to self-ligate badly. The vector : insert ratios I used for the ligation were: 1:3, 1:4, 1:5... Should I go further up? Any other ratio you would recommend? I CIPed the pBabe vector for 15, 30, 60, and 90 minutes, used above-mentioned ratios with all of them...nothing works.... the test plates don't look different from empty vector ones.... Any ideas????
Thanks a lot in advance!!!!! and thanks for your interest in the topic....
CIP and BAP are frequently problematic. The following protocol avoids the pitfalls common to these enzymes.
Most suppliers disagree on unit definitions. Be sure that you are using the definition the protocol or product data sheet requires. In addition, DNA concentrations determined by A260 may have 10-fold errors. Using a fluorometer and Hoeschst dye is much more accurate. Calculate the number of ends you are trying to dephosphorylate and dilute the CIP or BAP so that you are using exactly the number of units required.
Calculating moles of termini of double-stranded DNA
= 2x (g of DNA)/[molecular mass of DNA (Da)] or
= 2x (g of DNA)/[number of basepairs) x (660 Da/bp)]
CIP and BAP are among the most difficult enzymes to purify. Exonuclease activity is a problem when too much enzyme is used. The desired restriction site overhangs are easily nibbled away. Calculate pmoles of 5' termini accurately for optimal results. SAP may usually be used with less attention to this problem. However, if the 5' ends are recessed, the 37°C incubation utilized with SAP may not be as effective at dephosphorylation as 50°C incubation with BAP or CIP.
When dephosphorylating plasmid vectors for cloning, the lowest background of non-recombinants is obtained by using a precise quantity of BAP or CIP, terminating the reaction by phenol extraction and ethanol precipitation, followed by another dephosphorylation step and clean-up. Dephosphorylation is inhibited by phosphate, so single reactions may not go to completion. Excess enzyme will result in exonuclease problems. This technique avoids problems with nibbling of restriction sites.
Incubate at 50°C/60 min. Add 10 µl Dephosphorylation Stop Mix (80 mM EGTA, 4x TE, 40 mM NaCl and 2% SDS). Extract twice with Tris-buffered phenol:chloroform, once with chloroform, and ethanol precipitate with ammonium acetate and Linear PolyacrylAmide carrier. Because the CIP reaction is inhibited by the products, cloning vectors should be dephosphorylated twice with separate clean up steps to completely reduce the background.
I usually just put a unit or so of CIP directly into the restriction digest after it's complete, incubate for an additional 30 minutes at 37, and gel purify the whole thing. On the same gel (which contains guanosine as a UV protectant -- see here), I run out and recover my digested insert DNA.
Are you gel purifying your restricted and cipped vector?
I assume your XhoI - SalI insert contains no portion of your original vector? This could be a problem, if (for example) you're carrying over the ori from your original vector...
The vector : insert ratios I used for the ligation were: 1:3, 1:4, 1:5... Should I go further up? Any other ratio you would recommend? I CIPed the pBabe vector for 15, 30, 60, and 90 minutes, used above-mentioned ratios with all of them...nothing works.... the test plates don't look different from empty vector ones.... Any ideas????
I think you can go way up, the insert should not show up in the screening, this is evidenced by the vector only and vector + insert being the same... Add as much insert as your concentration will allow, you can even mix insert and vector together and co-precipitate before ligating to increase the concentration in the reacton, you could potentially go as high as 1:20 (maybe more but at some point you will have to transform so much DNA that the amount of DNA will affect the transformation efficiency....)
Especially if the insert likes to interact with itself you will need a concentrated reaction to force intermolecular interactions over intramolecular ones....
Thank you guys for all your advises.... I will try them all and then will update on how things go.... Thanks again...
hey you guys,
just as a follow-up of the topic..... Sal I digestion.... Are there any tricks if any on how to increase the efficacy of Sal I digestion (in particular, with Xho I).... I increased the time of restriction digest (RD), did RD overnight, increased the number of unit per reaction... Still my feeling is that Sal I doesn't cut the insert/vector properly that kills my ligation...
thanks