Dear all
I hope you are having a good time.
I am trying to ligate a PCR product of ~1500bp into a 6.5kbp vector (6.023kbp upon digestion). Somehow, I am not able to get colonies at all ! The digestion enzymes used are BamHI-Hf and XhoI, both are sourced from NEB. The PCR product was constructed through overlapping 4 different PCR products sequentially.
The digestion is performed as follows : 0.5uL of enzyme for roughly 1-2ug of vector @ 37degC. Heat Inactivation @65degC for 20 minutes and then gel extraction of the digested products.
Since nanodrop might give wrong values, I run 0.5 uL of the gel extracted products on the agarose gel to estimate the concentrations. I have attached the picture of the same. 1st well next to the ladder is that of gel-extracted double-digested vector while 3rd well from the ladder is the gel-extracted double-digested PCR product.
I used 5uL of the digested vector and 2uL of the 1500bp insert for ligation at 1:5 vector:insert ratio. However, I got only 1 single colony. I used the entire ligation mix for the transformation. As such I had also amplified and double-digested the inherent insert between the restriction sites in the vector itself (which was 600bp product. Upon digestion, it goes down to ~500 bp and I could see the 100bp fallout and the shift on the gel). Again, I used 5uL of the digested vector and 2uL of the 500bp insert for ligation.
As far as competent cells are concerned, I am using Stbl3 which are made chemically competent in-home. They seems to be working fine. I have also asked my friend to set up same reaction but with ligase buffer and ligase from their lab, which is from Thermo. Upon transformation of 5uL of that reaction, he could not see any colonies.
What I am planning further ?
1. In order to check ligase activity, I am planning to single digest the vector and then divide it into two : with ligase and without ligase.
2. I am going to screen this single colony that I got. In case it would be negative, I would set up a ligation at 1:20 vector:insert ratio.
I would like to ask you all what all can I do. Ligation is slowly becoming a serendipitous task for me.
I'd guess the issue is somewhere within the gel extraction.
1. If you're using UV illumination, the DNA might be getting too damaged for the plasmid to replicate once transformed. If this is the case make sure the transilluminator is set to longer wave-length UV at lower power then work as quickly as possible to minimize DNA damage. Another option is to switch to a fluorescent DNA dye which is excited by light in the visible spectrum like SYBR safe, but this requires a specialized transilluminator.
2. Most gel extraction methods require high concentrations of chaotropic salts to dissolve the agarose. If too much is left in the final product they can inhibit the ligase. These salts are why gel extraction products often have really bad 260/230 ratios. If you're using a column kit you can leave the de-salting wash buffer on the column for several minutes each time and maybe add an extra wash step. If you are using a kit, make sure it's pretty new (
Alexandra Johnson Thank you very much for your prompt response.
1. Even though I try to speed up the gel cutting process on the transilluminator, it could be that unfortunate cloning where I am getting issues with UV-damage to DNA. I will use longer wavelength UV next time, as you had suggested.
2. Even I have doubts regarding gel extraction. We use a gel extraction concentrate that needs to be diluted with 1/2 volume of isopropanol before we use it. I have seen it turning dark yellow/orange if we use bad quality isopropanol. As such, I have made sure that we keep them in the dark and use molecular grade isopropanol, sourced from Merck. As of now, it has not gone yellow. I had also tried Qiagen gel extraction (yellow) buffer but somehow even that has not worked for me.
In response to your suggestions, I will verify the 260/230 and 260/280 values for my digested vector and insert. I guess incubating with the wash buffer and/or washing twice might be a good idea. I would keep that in my mind for next time.
Also, since I had doubt on the ligase too, I have set up a following reaction. I have taken the same uncut vector and digested it for 1hr @37degC with HindIII. This digestion is expected to generate 5 fragments of varying length. After 1 hr, I inactivated it @ 80degC (as per NEB). Now, I used 5-5uL of this reaction mix to set up a self ligation reaction of 20uL in three replicates. In one tube, I did not add any ligase. In the other 2 tubes, I added ligase from two different sources. I am then incubating them at 25degC for 1 hr and then 16degC O/N. Tomorrow morning, I plan to run these three on the gel and see if I get any difference. The idea is that there should be size shift and change in intensity in the tubes where I have added the ligases (in this way we also bypass the gel extraction step so negative result could be correlated to the step with ligation per see).
I will keep you posted. Thank you again for your comments.
Hi and some tips:
1. I always treated the RE plasmid with cip.
2. tried to use the NEB T4 with high concentration: 2,000,000 units/ml
3. yes, I would add 1~2 ul RE plasmid and ~15 ul PCR insert for ligation. 16oC overnight.
Junjie Shao Thank you for your comments. Indeed, I use CIP in almost all my clonings. However, as I did not get any colonies in these ligations, I thought of skipping this step as sometimes CIP remains attached to DNA ends.
There is update on the experiment that I performed to check T4 DNA Ligase activity. A brief description : Digested the vector with HindIII to generate 5 fragments. Heat inactivated the reaction. Then split the reaction in 3 parts and using them set up ligations with or without ligase. I am attaching the picture of the agarose gel that I had run.
Alexandra Johnson I think that gel extraction process could be the problem. I am yet to check the 260/230 and 260/280 ratio as of now.
Hi everyone.
I performed the digestions again. I carefully gel purified the digested vector and insert, performed 2 Ethanol-buffer washing with 5 minutes incubation at each step.
I loaded 1uL on gel to check the concentration (photo attached).
I had then setup the ligation with 2uL of digested vector and 4uL of the digested insert @16degC for 16hrs. Again, I did not get any colony.
Then, I have performed only vector, only insert and vector+insert ligations @25degC for 1 hr and then loaded on the gel. (photo attached). It appears to me that the insert has been digested and getting ligated to form concatamers. Somehow, I am left clueless as to what should I do next?
Hi Abhishek,
At first glance there are a couple of items that could be the cause of your unsuccessful cloning.
> Partial digestion of insert by the enzymes: Did you check that the number of bases flanking the sites are sufficient for an efficient activity? (check attached file1)
> High UV radiation: Alexandra well pointed out above that this is a critical point. In fact, after I switched from UV to a blue light source my cloning efficacy improved substantially.
> Saturation with insert: Estimating insert concentration by intensity in the gel is quite tricky since it doesn't have in account that smaller molecules have a higher effective concentration than larger ones. Let's call it a higher effective concentration of cohesive ends. Please use the excel sheet I attached (File2). Trust your nanodrop (make duplicate/triplicate measures) and insert the values in the sheet. I made so you get the values directly on the righthand side of the sheet. I use 0.015pmol range (first tab) because I use homemade ultracompetent bacteria (Inue method, see attached File3). I will test 0.015 pmol and 0.15 pmol range in vector:insert ratios from 1:1, 1:2 and 1:3. In my experience it doesn’t make sense to go into higher insert ratios.
> Inefficient competent bacteria: I have typically bad experience with Stbl2 and Stbl3 strains when I have to transform ligations even when prepared as ultracompetent. Unless you specifically need them (ITR-Viral constructs, unstable or extremely large plasmids, etc) I wouldn’t recommend them at all for regular transformation with ligations. If you need these types of special bacteria I strongly recommend “NEB stable, cat# C3040H”. They do work well and prepared as ultracompetent they are very efficient for transforming with ligations.
What to try next:
- Cut you plasmid and insert with the 2 enzymes sequentially, specially the insert, with BamHI at the last. I like better overnight digestions.
- Run a little fraction of the digested vector/plasmids in a well of the gel and the rest in the following gel. Then go to your UV source and place a piece of aluminum foil so it protects the second -more loaded- well from the UV. Cut out the band in the other less loaded well which you’ll use as reference to blind-cut the other band without UV exposure. After cutting, you can check how god you did under UV.
- Purify and quantify vector and insert. Use the excel sheet to get the amounts for the reaction. I like using 0.5 ul T4 5U/ul (Thermofisher cat# EL0011) every 10 ul of reaction. Maybe an overkill but it works every time so I won’t change it. Again, try 0.015 pmol and 0.15 pmol range in vector:insert ratios from 1:1, 1:2 and 1:3.
- Transform
What to do if the last doesn’t work:
Seriously think to move into cloning techniques based in overhang homology (homologous recombination) like Gibson assembly and SLIC (File4)
https://blog.addgene.org/plasmids-101-sequence-and-ligation-independent-cloning; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668909/
https://www.addgene.org/protocols/gibson-assembly/
These are extremely efficient cloning methods and once you buy some basic reactives (which you may already have) your only expense will be on primers. In the long run it is way cheaper than regular cloning.
Another advice, use terrific broth (http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8620) instead of LB. Colony growth rate is almost twice as faster as with LB and plasmids yields can be really incredible compare to those using LB in the same time frame.
Although nice every time to time, do not let serendipity to rule your life. Check your protocols ;)
Cheers,
Dear Sebastián Dupraz
Thank you for such a detailed response.
I would like to share some of the results of the troubleshooting that I did.
1. In order to check whether each of the enzyme, BamHI and XhoI, digest the insert or not (for vector I was sure as I could see the fallout), I did the following experiment.
a. First, I took the undigested insert, added ligase and kept it at 25degC for 1 hr. Then, loaded on the gel. (Please refer to Fig (A)).
As we could see, there is no difference between -ligase and +ligase. This is actually obvious and what one would expect.
b. In order to check single restriction digestion efficiency, in one tube I digested the insert with BamHI and in other tube I digested with XhoI. The digestions were performed for 1 hr at 37degC and then heat inactivated at 65degC for 20 minutes.
Then, I added ligase and buffer to each of these tube to make up a 20uL reaction. We expect to see formation of 3000bp apart from the 1500bp due to self ligation. However, we do not expect to see formation of 4500bp and so on because this is not double digested insert.
(Please refer to Fig(B)) As we could see, both the digestions were working. However, XhoI digestion seems not be working as efficiently as BamHI. This could be because we did not have XhoI Hf version as opposed to BamHI, which we had in Hf version.
c. To finally confirm double digestion, as I did earlier, we could see concatamer formation in the tube where the double digested insert and ligase was added.
Summary as of now : BamHI and XhoI digestions for insert are working, even though XhoI is not cutting as robustly as BamHI.
- I had again set up the ligations with different amount of vector and insert. I also used Top10 apart from Stbl3 for transformation of half of the ligation reaction in one case.
- Surprisingly, I got around 12 colonies in Top10 transformation but when the same amount of ligation mix was transformed into Stbl3, I did not get any ligation. This potentially indicates at incompetency of Stbl3 compared to Top10. Even though my ligation was not working that efficiently, but relative poor competency of Stbl3 made the task more tricky.
- However, to add to the twist, my vector is actually a lentiviral transfer vector and therefore has high propensity to undergo recombination in Top10. I am growing the 12 transformants that I got at 28degC to minimize chances of recombination. I will let you know the results of this experiment.
Sebastián Dupraz Have you ever tried preparing electrocompetent Stbl3 ? I was thinking if electrocompetent Stbl3 cells could be made as they would have higher efficiency compared to chemical competent Stbl3.
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