My cloning experiments are failing and I do not know which step is the problem, though I am suspecting the ligation step.
Everyone has suggested something that is not wrong in their perspective. Yes, you can check by the suggested means but none is too easy.
You have written that your cloning is failing. It would be better to specify: whether you are getting colonies or not. If you are not getting colonies, then the failure of ligation may be a possibility. In that case, to check whether the ligation reaction is running fine or not, I will suggest you to use a ligation positive control such as the empty vector digested with a single restriction enzyme (RE). If you are able to get colonies of self-ligated plasmid then ligation may not be a problem.
On the other hand if you are getting colonies but none of them are recombinant, then there can be several problems, which may be solved as follows:
a. If you are ligating doubly digested DNAs then one of the two restriction enzymes is not digesting completely - Ensure complete digestion by prolonged incubation and/or increasing the enzyme concentration. If you are using an RE that shows star activity, then incubate with the other enzyme first for sufficiently longer time then with the second.
b. Use varying molar ratios of vector and insert ranging from 1:1 to 1:5. Using higher insert (1:5) works fine, almost always. Use gel based eye-estimation.
c. Take proper precautions while gel purification, if the insert size is very high (say >2 kb) or very small (say
as far as I know you can simply load a part of your ligation-reaction on a gel. At least this works for my ligations. The size-change will tell you whether the ligation was successful
cheers armin
What about good old re-ligation control? Apart from your regular ligation reaction, you mix a separate reaction with all chemicals but the insert. You treat and transform this re-ligation control the same way as your ligation. The amount of colonies on control plate tells you the frequencey of re-ligation of your vector backbone to itself. U can then roughly predict what is the ratio of successfully ligated colonies vs. failures. If the control plate is as full of colonies as your ligation plate, it is futile to start preps on those. U can also try to de-phosphorylate ends of your vector with CIP to prevent re-ligation (http://www.neb.com/nebecomm/products/faqproductM0290.asp).
Good luck
Re-ligation control was done, but there were no colonies on the plate.
Just to make you aware... the other way is to do resriction digestion of your ligation product.. If you get band at your gene size you can cofirm the ligation success. But yes there is dawback of loosing time and ligation product both, hence it is not recommended !!!
Try including a positive control of ligation (vector digested with one enzyme, gel purified, and re-ligated). If that gives you colonies (and the same reaction mix without ligase does not), your DNA ligase is working. Does your insert come from a PCR reaction? It could be that the one or both restriction enzymes are only partially efficient on the amplicon.
>Is there a way to check for the success of ligation experiment before transformation (and without running a PCR)?
I guess you should run a PCR to check your ligation.
You can run your linear vector and ligated one on the agarose gel by appliying electrophoresis. Due to their masses are different you can easily identify the one that you ligate..
Did you check if your cells are still competent? I mean, in paralel, have you tried to transform the competent cells used with another plasmid ? Good luck :-) !
Hi Nicolas............
I believe running a PCR will not confirm the ligation because if template is not ligated, still then you will get the desired amplification.
@Arun
of course you'll have to use correct primers for that, e.g. ligation-spanning primers (1 primer on vector, 1 primer on insert). if you use 2 primers which are located on your insert and directed to one another it obviously wont make a whole lot of sense.
however, if you choose correct primers you should be able to confirm your ligation
Doing restriction digestion (RD) as suggested by others doesn't seems to be a good idea. I disagree with the idea of RD followed by gel electrophoresis. Think of quantity of vector required for RD and visualization in a gel. It will be too costly affair....
I suggest to follow the normal procedure only. Make sure you are doing every step perfectly. Ckeck transformation efficiency of your competent cells using some positive control.
What kind of vector are you using? Is it TA cloning or what. If it is TA cloning make sure, you are using right kind of Taq-DNA Polymerase. Some of the high fidelity Taq-Pols have 3′-5′ exonuclease activity and hence generates blunt-ended PCR fragments. You can go for A-tailing of your PCR product. Purify the PCR product or run in the gel and extract the desired amplicon. Go for quantification. and ligate in proper ratio. Its better to proceed with good quality competent cells.
You need to stop purify your vector in agarose gel.
Only process l with sap after digestion.
You need to ensure that your cells are competent before you proceed towards transformation. One can go for restriction digestion or run a gel, whichever suits your experiment. But, as others have pointed out, you will be compromising with the quantity of your ligation product, if any.
I would suggest you to standardize the protocol that gives you successful ligation and then, follow that precisely. Good luck with your experiments.
Everyone has suggested something that is not wrong in their perspective. Yes, you can check by the suggested means but none is too easy.
You have written that your cloning is failing. It would be better to specify: whether you are getting colonies or not. If you are not getting colonies, then the failure of ligation may be a possibility. In that case, to check whether the ligation reaction is running fine or not, I will suggest you to use a ligation positive control such as the empty vector digested with a single restriction enzyme (RE). If you are able to get colonies of self-ligated plasmid then ligation may not be a problem.
On the other hand if you are getting colonies but none of them are recombinant, then there can be several problems, which may be solved as follows:
a. If you are ligating doubly digested DNAs then one of the two restriction enzymes is not digesting completely - Ensure complete digestion by prolonged incubation and/or increasing the enzyme concentration. If you are using an RE that shows star activity, then incubate with the other enzyme first for sufficiently longer time then with the second.
b. Use varying molar ratios of vector and insert ranging from 1:1 to 1:5. Using higher insert (1:5) works fine, almost always. Use gel based eye-estimation.
c. Take proper precautions while gel purification, if the insert size is very high (say >2 kb) or very small (say
@Amit
I am ligating double digested DNA. A gel is run and the product cleaned, before ligation. A posiive control is always included, and there are no colonies on the plate.
Thank you all for your suggestions
>Hi Nicolas
>I believe running a PCR will not confirm the ligation because if template is not ligated, still then you will get the desired amplification.
Hi,
As mentioned one primer in vector (e.g. forward) and one primer in insert (e.g. reverse). You should have amplification only if the ligation worked.
>I am ligating double digested DNA. A gel is run and the product cleaned, before ligation. A posiive control is always included, and there are no colonies on the plate.
How many times did you perform the ligation? As mentioned before you should try again from the beginning with adequate control in each step.
If you are not getting colonies of self-ligated vector (as mentioned in my earlier post), then please change the ligase and ligase buffer.
Good Luck!
Hi, the suggestion I will make sometimes works because is difficult to visualize. It is to run your ligation in an agarose gel. Doing this you may see bands with different sizes, corresponding to your insert, your vector, the ligation product (Vector+insert), and you may see monomers, dimers or even trimers of your insert or vector. I have done this and it works, sometimes...
I hope this this can help.
You have got many suggestions, and most of them you must have tried( among these, I prefer running products on gel).
But I will suggest a simple thing here which I have tried once and worked for me. Ligation not working with no colonies and you tried many things. Just double the amount of vector and corresponding ratio of insert, and reduce the volume of ligation reaction by half. Keep the ligation overnight at low temp., I am hopeful that it will work (assuming that your buffers and enzymes are working, which you obviously had cross confirmed in response to above said suggestions)
Another thing to add which might be the problem. your restriction digestion, sometimes it doesn't happen, so just cut any standard vector and see. And one more thing if you are digesting the PCR product , then I hope that you have left enough bases after RE site for enzyme to work on it.
@ Armin
You are absolutely right....Vector specific primer pair or alternately one gene specific and vector specific primer pair will do the job....
I normally check the ligation by gel electroporation. In some case, if there are too many ladder-like bands, pre-treat the ligation product with plasmid safe nuclease. You will be able to see it on the gel.
Trouble shoot each step of the procedure. First run a gel to confirm ligation. Run positive and negative ligation controls on same gel to confirm they are clean and correct. If everything looks good there than it might be your cells. Run a positive control transformation using 1ng and 100pg plasmid DNA. Plate on antibiotic media and count cells to determine if the transformation efficiency of the cells is high (10-8). Lower efficiency((10-6) could mean that you need new cells, need to plate entire transformation, need longer growth of cells, etc. Also using 2 different quantities of plasmids will give you a sense of how much ligation you need to add to the transformation.
normally cloning failure can be due to many reasons
1. first and foremost is your competent cells. check whether they are competent enough.
2. then come to your digestion, exonuclease contamination can also lead to failure as it chop off your overhang. so maintain dna in 0.1X TE buffer
3. then come to ligation please check your ligase using lambda HindIII marker, whether it is working or not. change your buffer or add fresh ATP to your ligation mix
4. last thing which is most unusual is to find whether your gene is toxic to the bacteria or not.
these things has to be checked irrespective of single or double digestion
vector to insert ratio as far as i know it doesn't matter
you can chek by loding 2 to 3 ul of of your ligation product on gel. If you will get plasmid form your ligation has occured. If you are facing problem with ligation, use gateway cloning technology where you don't be having restriction and ligation.
If you were my student, I would have you run the PCR and do a digestion. In molecular biology every step is important and needs to be as perfect as you can get it before proceeding. Getting a ligation wrong or in the reverse orientation can cost you a lot of time and money.
Best you run ligation product on agarose gel.......it will give you a clear idea about ligation
Nobody has mention this, I guess we all assume that the insert and vector were prepared correctly. I would also check the beginning of everything, such as your PCR design. If you have a "wrong" enzyme site designed in your cloning PCR primer; or whether you have extra bases to protect your enzyme sites? did you run a gel to check your PCR products and digested vector? Because a lot of time NanoDrop type of measurement of DNA concentration might not give you a accurate estimate. etc.
There are so many factors that can derail your final cloning effort. You really need to be careful and systematic on each and every step of the process! Good luck!
You might have known about the vector size and all before ligation. so running the gel is the best one to get to know about the progress. and one more thing is you should check about your enzyme restriction sites and target gene cutting sites also. if it were in wrong you can't get the product. did you run the gel to check your products before and after ligation process?
As per kejun Guo's words so many are there to consider still. So, my suggestion is check and get therotical result about each and every step then you will get it........
Cheers Dude......
it works for me working with competents cells prepared at the moment, it takes two hours and gives good eficiency. Then check every step with appropiate controls..and sequence the ligation to know if there were no wrong protein produced due to worng primer sequence (once happened to me).Good luck
i usually like running a ligation product on a agarose gel prior to transforming it into E. coli cells. As a control, I like running on the side each of the ligation components, if be a vector, i like to also run the insert and the backbone in the lane afterwards to show a difference as a control.
Find a restriction site that is unique in the insert and digest your ligation product . Then run on a agar gel this should give you an idéa but that dépend also on the size of the insert.
I have to say that if your strategy is good, and you can see clearly your DNA in a gel before ligation, it should work. If none of these are the problem, check your enzyme (they tend to degrade very fast). The key here is not to check the product of a ligation beforehand but to be sure the cloning strategy is correct. For that, check primers (if used), check restriction enzyme activities, check vector/insert ratio, check phosphatase (if used).
Based on your strategy and the controls you can figure out where is the problem. Do you have no clones? Do you have re-ligation of the vector?
If you suspect the ligation efficiency, performing a parallel reaction with ligase from another laboratory could be a way to check that your ligase is still active.
I am agree with Vinod Sangwan, you can run your ligation reaction in a gel compared the circular DNA, linearized DNA plasmid, pcr product alone before your ligation, and the ligation, then you can compare the difference DNA in your gel and make an approximation of how long in base pares is the recombinant plasmid that your hope , I mean linerized plasmid plus your insert, but in any case you have to make the transformation and then the mini preps and the sequence analysis to be sure about your recombinant plasmid. Be sure the ligation with relation 10:1 plasmid:insert in minimum volume. You can make your competent cells by Calcium chloride Protocol 25 Molecular cloning book. Method modified by Cohen and Cols. At the end of the protocol make your own aliquots with 50 ul.instead of 200 and they are enough to transform cells and you can keep this at-80 ªC for a long time and save money . Good look
Yes. Comparing equal aliquots of the ligation mix taken at the beginning and the end of the ligation reaction by agarose gel electrophoresis will help, if you have enough DNA. The presence of high molecular weight molecules after incubation will be indicative of successful ligation.
If your insert has ligated to the backbone, then you need to cross check with insert release and see that your insert and vector are released in the same size range as you would know. The insert release should be with the same RE you used for your digestion. You can refer to Molecular cloning by Sambrook, the best reference for such checks. Since there are sometimes chances of self ligation you need to check gels and see for the double digestion.
simply run agarose gel of the ligation product using DNA to which you want to ligate your desire DNA as a control. if there is a band is similar in size of DNA then there is no ligation and if ligation product shows higher molecular band then DNA so there is ligation occur...
Run 1/10 of the ligation on a gel, if you use as controls the vector alone, the insert and the vector re-ligated you should be able to see the ligation products.
nowadays you can se bands that correspond to 5 ng of DNA esasily. You know what you aim at.
Restriction digestion of the little ligated product with the same restriction enzyme as used of cutting plasmid and DNA of insert ....... and running on a agarose gel electrophoresis and you should get two band one of vector and another of insert......
If you are ligating the partial digested product to the backbone. this type of ligation reaction cannot be confirmed by agarose gel electrophoresis. I think it is better for you to set another ligation reaction that have only backbone ( in same amount that ligation mixture of both backbone and insert have). transformed both the mixture to competent cells in two separate tubes that must have been prepared in same batch. check the transformation result in both case if you are not getting the clones in case of ligation mixture that had only backbone (control) then you could able to say there was a problem in ligation specially w.r.t ligase . if you are getting good efficiency in case of control and you are not getting the clones in case of ligation mixture that had both backbone and insert or you are getting the very few no. of clones then you should go for the an optimization that will tell you the best backbone insert ratio which leads to increase in the transformation efficinciey. you should have to also take care of size of the recombinant plasmid that would form after the ligation reaction........ All the best for your ligation and furthe r steps..
You may run the ligation product on the gel to see if it worked. You should see multiple bands of higher molecular weight than your empty vector, as well as a band of the same size of the insert, since you should be using an excess of insert. Also, before ligating you should smell the ligase buffer. It seems weird, but if it doesn't stink to sulfur, it means the DTT is gone and that the buffer is not working anymore. In our lab we dephosphorylate the vector and use phosphorylated insert. It helps with the efficiency.
Hi all,
I also have faced a difficulty of ligation recently.
I checked restriction enzymes I used were active, and competent cells still maintained their competency. So I doubt ligation was wrong.
This thread is what I'm looking for.
As most of people here says, running the ligates on agarose gel seems to be simplest way to comfirm the ligation goes well.
I have a question further about it.
What's going on with ligates in agarose gel?
I put my gel image. Both ends are markers. Lining ligation (left) and insert (right, 0.5-0.9kb) side by side except rightmost lane, which is the linearized vector plasmid (3kb).
DNA was cut by EcoRI and XbaI, so both ends of each fragment have different sticky ends.
The amount of ligates on gel should be estimated a half of the insert and vector DNA in next lanes.
Since this trafos failed, I didnt expect I could see desired bands.
However, the result was somewhat weird.
I consider the slowest band is tandemly ligated vector dna.
Why does it occur in spite of the insert-vector ligation?
Can I expect to see strong ligated dna if ligation works well?
Doesn't ligated DNA run lower than expected? For example a 4000bp plasmid will run at 4000 when it is linearized but sometimes up to half that size when it is ligated.
Treating your ligation productions by restriction again (enzyme site not on your target gene).
@kenzi u had the procedure done well, but perhaps you didnt optimize youtr ligation mix. What was the ratio of your buffer to the restriction enzymes and to the plasmid? The buffer, enzymes, insert and plasmid should be about 25:1:1:1. If it was so then perhaps you place more EcoRI enzymes than XbaI endonuclease. I have not worked with XBaI but Hind III and I had to put a ratio of EcoRI to HindIII 1:3 respectively. I hope this helps. @souza as mentioned above u can obtain your plasmid from the transformed cells and run on agarose gel, with your uncut plasmid as your control. If similar bands are present then your ligation was unsuccesful. Ur band should be formed slightly above the uncut plasmid control. Cheers!
I agree with Vinod & Richa. Run the vector, insert and the ligation reaction. If the ligation reaction is limiting, you may want to increase the amount of ethidium bromide in the gel.
Dziedzom, you mentioned that you are not sure about which step of the cloning is failing, further, please mention why you think that the ligation step is a problem. Is it because you don't get colonies out the ligated product- transformed cells or you get false positive colonies everytime you do the cloning? Please mention exactly and I would try to help you regarding this. Also, make sure that the cloning steps before ligation have been correctly done, run agarose gels each time to check for PCR amplificatio/restriction digestion and use positive and negative controls each time. Thanks.
In my opinion, the critical step (I would say 80%) for a sucessful cloning is you to have a competent bacteria above 10e7. Whenever you carry out the ligations of your inserts, you should also include a ligation of a vector only (with both ends compatible).
Dear for checking ligation plz go for the tedious process of transformation and screening by colony pcr. Running agarose gel for confirmation will not give you clue of ligation to much extent. you can use ATP during ligation as the ATP tend to degrade over repeated freezing and thawing. Also try to Alkaline phosphatase after digesting vector as you skip this step......vector tend to religate. While clonning you should be sure of every buffer even the quality of water you are using always put only vector ligation mix without insert for vector background. for further query mail me to [email protected]
For cloning trouble...
1. I agree with Anil who suggests phosphatase treating the vector.
2. Note that some restriction enzymes do NOT cleave efficiently near the ends of DNA: for PCR products you may want to add 2-5 nt between the end and the restriction site to ensure efficient cleavage. Really old NEB catalogs have this information in a chart with enzymes and the seq/length required for efficient cleavage.
If you are cleaving a vector, this may mean that in a sequential digestion, you should use the enzyme that is sensitive to ends first on the intact plasmid, followed by the second.
3. T4 DNA ligase has both ATP and DTT in the buffer. These components will go bad first if it has been freeze/thawed many times or is very old. You can make your own buffer (manufacturers list the components) with fresh ATP and DTT to help your enzyme work well.
Dear Dziedzom, Run the ligation mixture, digested vector and target gene on agarose gel. You will find the decreased electrophoretic mobility of ligated gene. If ligation is complete you will not find any band of gene size.
Always try to quantify your experiment, I always use insert:vector by 3:1, and I never check the ligation rate and I always get what i expected.
Hey check your comp cells because sometimes they also cause a problem did you check the transformation efficiency.
I think you can keep a positive control (which will definitely show colonies as for example pGLOW) side by side your unknown experimental sample during the transformation then at least you will know that nothing is wrong with the transformation step.
Usually different ratios of insert to vector is used in case of ligations (which depends on the concentration of both). Ligation reactions are also done using various conditions as at room temperature or at 4 oC for 24 hours. Different conditions can be used.
Using different insert to vector ratios, different ligation condition can be helpful.
All the best,
Mithu
Add one extra step prior you run your ligation on a agarose gel, treat your ligation product with 1uL plasmid safe DNA nuclease, which removes all linear fragments but not the circular plasmid. You will be able to see you plasmids, with correct size appeared if the ligation is successful. Best of luck.
You can use TempliPhi from GE Lifesciences that only amplifies Circular template.
The best sensitive way is to use Agilent Bioanalyzer 2100 using DNA High sensitivity kit. It will give you very sensitive results.
Well, the gold standard is to run qPCR to assess this ligation. There are different kits in the market available for Ion Torrent platform. The cheapest way (in my opinion)
is to assess the size of the fragmented DNA and then the ligation using the Bioanalyzer
yes, when you do the ligation reaction, prepare additional tube. use one to do the transformation. and the other tube to run on gel. you should run on the gel 5 lanes: 1-DNA marker ladder, 2-undigested vector, 3-digested vector, 4- the insert DNA that you wish to ligate to the vector, and 5- your ligation tube. if the ligation was successful, you will see the size of the ligated tube, is higher than all other lanes . and confirm in the same time that the vector is fully digested or not. your problem in ligation is usually not the digestion or the transformation, because, if one copy is successfully cut, or introduced to a bacterial cell, it will produce a colony. the critical part is the de-phsophorelation step. focus on this, and i usually make my ligation total reaction as 10 ul and also the dephosphorelation as well. if you made it more than 110-20 ul total volum, the risk is getting higher to lose your ligation reaction.
as i commented in another forum, add fresh ATP into ligation buffer from a reliable new ATP stock, to final 1mM. ATP is the 1st component, which goes bad w/o warning in ligation buffer, especially if it has been frozen-thawed many many times
If you are sure that your are not getting results just bcoz of your ligation, then run your product on electrophoresis gel. Other wise send me your ligation protocol and concentration of your DNA
there were good suggestions. What i believe was not mentioned is what to expect when running ligation rxn, vector alone, and insert alone. In "ligation rxn" lane you should expect to see multiple bands, not just 2 (one for insert and one for vector). There will be insert+vector, insert +1-2=3 inserts, there will be circular vector (if same sticky ends were used w/o dephosphorylation). If DNA conc. is low, overstain your gel with EtBr, wash 5-10 min in water, and take a long exposure. Absence of any other bands (rather than vector, insert) will tell you that indeed ligation is not working at all.
Running a gel is an alternative if you don't want to run PCR, but you can't cut corners or you won't be able to understand the results, and that means that you can't run superquick gel protocols. Also: run a preligation control (an aliquot of your ligation mix with everything but the ligase) in the lane by the side - plus pretty much every other control you can think of. Ligation gels can be pretty tough to interpret!
Can't you just use the blue colonies formation system?
Half an hour before plating your cells, prepare the plate with IPTG/x-gal. The blue colonies will be the colonies formed with cells that did not recombine with your DNA while the white colonies are the good ones. It saved my life when I did my gargantuan screenings. I used the Promega pGEM T-easy vector system.
I didn't remember the mechanism completely so here goes a copy/paste of it:
"The blue/white screening method works by disrupting this α-complementation process. The plasmid carries within the lacZα sequence an internal multiple cloning site (MCS). This MCS within the lacZα sequence can be cut by restriction enzymes so that the foreign DNA may be inserted within the lacZα gene, thereby disrupting the gene and thus production of α-peptide. Consequently, in cells containing the plasmid with an insert, no functional β-galactosidase may be formed."
It's really easy but you must be sure that your plasmid carries the lacZ.
Hello Dear as Mr. Vinod told u that run the ligated product on agarose gel, if you will find smear on the gel then your ligation is ok. Thanks
Dear you should run ligation product on agarose gel electrophoreses......
Hello dear, why you runaway from PCR, simply run a PCR and confirm ligation, it is the best method.
If you are not going to do transformation and PCR the quick way to determine that you ligated successfully your insert into vector is taking ligation product and run it on Gel. Make one addtional tube of ligation reaction and on agarose gel run following- 1- marker 2- Vector undigested 3- vector Linearized by single digestion 4- your ligation product 5- your ligation product with single digestion. .
It may be possible that circular plasmid vector/ ligation product give unusual migration over gel so single digestion will be good. which will give you idea of exact size of your ligation product. if its equal or near equal to vector size (Kb)+Insert (Kb/Bp), your ligation is successful.
Can you give reasons for suspecting ligation. What if the competent cells are bad or your PCR product is of poor quality
Why You want to check the ligation?
I dont know what kind of step you are following but I can suggest you that. once you just try this modification may be you will get...
1. When you are keeping ligation so just add your full gel extract. don't add water.
2. When you are going for transformation that time if you are making competence cell so just give liq Nitrogen step then after taking out immediately add your ligation mixture. or if you are using commercially available competence cell, in freeze state only add ligation mix don't allow your competence cell to thaw.
I think some one already mentioned to select proper controls so to identify the root cause of which step failed.
And it would be great if some one could list essential controls which we must take into consideration while from the start until end during construct cloning.
Cheers
A
This is my standard list of controls:
Uncut vector
Cut vector in ligation buffer, but not ligated.
Cut vector, ligated.
Cut insert + vector, unligated.
Cut insert + vector, ligated.
Uncut vector + cut insert, ligated.
Insert alone, ligated.
I think that covers just about everything, but if you can think of another control, you probably need that as well!
So my understanding is
Uncut vector with just water ---- to detect transformation/ abs and comp cells probs
Cut vector without ligase interprets for undigested vector
Cut vector with ligase interprets for undigested and relegated vector
Cut insert +cut vector without ligase ????
Cut insert + cut vector with ligase --- normal ligation reaction
Uncut vector + cut insert, ligated. ???
Insert alone, ligated. ????
That is absolutely right that almost evthng is covered but do we really need too many controls ??
A lot of factors are involved in failed cloning experiments. First of all you need to verify the transformation efficiency of your competent cells using undigested plasmid of known size. Then you should always perform transfomation alongside with controls (most of which have been stated above). The choice of the enzymes, buffer compatibility and their proximity in the MCS of the vector also affect the digestion efficiency. When ever possible, use enzymes that are far apart in the MCS of the vector. If no distant enzyme sites can be used for your experiment, you may wish to try sequential digestion. The ligation itself can be performed using different molar ratios of insert to vector. In most cases an insert to vector ratio of 3:1 to 5:1 has given me good results. Next, Try to set up ligation reaction on ice and depending on the size of gene, you can prolong the ligation incubation step. For instance, I use up to 30 min incubation when cloning a gene of about 6-7kb using thermoscientific kit. I believe if you watch your controls very well and optimise some parameters, you will get positive clones.
Best of luck
Bashir
Dear you should run ligation product on agarose gel electrophoreses....
If you know the sequences that are flanking the insert region (the portion on the vector) you can perform PCR on the ligation reaction. You will receive a product that is similar to your insert length plus the primer region. You only need a low quantity of material for amplification to be successful (~25-50ng/ul) depending upon how specific your primers are.
I suggest you run your ligation products on agarose gel alongside the control (vector only). you may also want to use pcr to screen for the insert; in this case use a primer that was originally used to create the insert and one that binds to the vector.
In my experience and modicum of knowledge, you can run the ligation product on an Agarose along with plasmid. You would probably see a slight different migration of the products as compared to control plasmid under the electrophoretic field.
I´m agree with Tripathi, you can make a mini preps from 10 o more colonies to get the DNA, then make a digestion with the selected restriction enzyme, calculate the size that you will obtain with and without insert, run all the digested DNA from the colonies in a agarose gel together with the linearized vector and select which of them had the insert. I suggest use ratio 10:1 insert :vector for the ligation. Good look!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology