I am trying to clone two genes on one single vector. One gene is 1800 bp and the other 2900 bp. My vector is 10 kb ( pBIN Vector) and doesn't have my desired restriction sites so I designed primers containing leader sequence, my desired restriction sites and part of the gene sequence. I have considered BamHI and EcoRI restriction sites for one gene and EcoRI and SacI for the other one. My vector has BamHI and SacI restriction sites. I amplified both genes using proofreading DNA polymerase, Hercules II fusion from Agilent Technology, USA. I got the right size of the genes on the gel then gel purified DNA, digested with appropriate enzymes, running on the gel again, still everything was fine. I Ligated two gene fragments with T4 ligase, running the product on the gel and I got only one band around 5000 bp confirming the ligation of the two genes. The problem starts when I want to clone the ligated genes into the vector. I have tried regular ligation including vector digestion and ligation of vector and genes many times. I am getting colonies on the plate. I tested colonies with PCR. I got bands then I grew them in LB with antibiotic. They grew but when I was trying to extract plasmids the yield was so low around 20-30 ng ( i usually get 150 ng for this plasmid). I digested again but i was only see the back bone and no insert. I also sent them for sequencing, results were negative. I also have tried TA/ TOPO cloning, the same results were obtained. I have done many cloning before but not by PCR. All the kits and enzymes are fine since I am using them all the time. I have tried any method that I could have think of, none worked. I didn't use Taq polymerase because of its high rate of mutation but now I am going to try that one too to see if the problem is with Herculase. This is while ligation at EcoRI site worked. Now I am wondering if anyone can give me more advise about what I have done wrong or what I can do to solve this problem.
Hey there,
Cloning by PCR technique is relatively easier than other methods. From your query what I understood is that Ligation is working fine, the only problem is downstream. How sure are you about your vector backbone not getting self ligated? Moreover have you tried for alkaline phosphatase treatment to prevent vector from self ligating at the first place itself? I fear that your vector might be self ligating, unless you are sure or have included proper ligation controls for the ligation reaction with vector.
Cheers.
As you are getting PCR products from the colonies obtained after transformation, your vector-insert ligation and transformation is quite fine. Now my question is-
Did you use the same colony for inoculating LB-amp with which you got PCR product?
If you did so, there should not be anymore complexities.
If not, you should follow a slightly different method. Pick the colonies you see after transformation to inoculate LB-amp broth. After overnight incubation, use 1 µl of the culture from each tube for PCR detection. Store the culture in 15% Glycerol at -80 which are positive for PCR. Subculture them in LB-amp agar and extract plasmid from them. Use your plasmid for PCR, sequencing and digestion.
The answer of following this is, not all the colonies you see after transformation carry your recombinant vector. During digestion, few percent of the vectors remain undigested. So, finding PCR products with few colonies doesn't imply all colonies to carry the desired recombinant vector. Hence, the above mentioned lengthy procedure is to ensure that you do not loose your colonies with recombinant vector.
A couple of suggestions : After your first ligation of the 2 genes, have you tried a PCR amplification on a small aliquot of the ligation with the BamH1 and Sac1 containing primers (eg use 1ul of 1/10-1/1000 dilution). This will confirm that the primary ligation has taken place. How far from the termini are your restrictions sites - do you have to have enough extra bases to ensure efficient cutting?. If your product is confirmed, cut and proceed withe the ligation into vector. I might be tempted to try a very simple PEG pptn to get rid of primers / cleanup. Problems using gel purified DNA in cloning are not unknown.
After the second insert + vector ligation do the PCR check again to demonstrate that you are getting ligation using M13 for-rev primers. If you get nothing then there is a problem with either the RE cutting or the ligation. As another possibility you may consider cloning the PCR product 1 directly into a "cloning plasmid" eg pJET. This will ensure that you have a large amt of starting material to manipulate before putting into a shuttle vector
Dear Tasaduq. To answer ur question I have done many cloning with this vector no problem before. I have also used alkaline phosphatase . That's not the case 😔
Dear Asaduzzaman, I have exactly used the same colony. I grew only positive colonies. Still no results after sequencing.
Thanks Ian, very helpful comments. I will try them all. U cheer me up. Thanks 😄
Dear Homa,
I also thing as Teo above, that the problem might be restriction or ligation failure. So:
- Have you done a gel extraction after digesting your insert with BamHI/ Sac? If so, how was the purity of the extract - if very low, the ligation could fail.
- Have you tried to force the cloning by using higher insert molar concentration? I know it's basic stuff, but just in case :)
- Have you tried to include positive ligation control in parallel - e.g. to clone a known insert with BamHI/ SacI sites in your vector to ensure that the ligation works? If not, you could try and if everything is fine, it is probably the restriction. Then, verify your primer sequences and the restriction enzymes with proper controls.
I hope these will help you. Good luck!
Hi Homa!
I would try the following:
Have both inserts (i1 and i2) purified in different tubes AFTER digestion with corresponding enzymes and in a third tube have purified, digested/dephosphorylated vector (v) (although considering that you use different enzymes to open the plasmid, dephos. is not mandatory. However, in case you have one enzyme working slowlier than the other, you may have a fraction of single digested plasmids that could re-circle)
Then, I would make the following T4 reactions: (v+0), (v+i1), (v+i2) and (v+i1+i2). Another tube will consist only in v-t4 (and no inserts), keeping always volumes and molarities identical. Finally transform the 5 rxns and streak plates.
From this, you should get a significant larger number of colonies in (v+i1+i2) than in the rest of the cases, and also you will have a clear idea of the backgraound of false positives at the time of picking colonies for testing using Bam+Sac upon miniprep, or by "colony PCR" using F primer sitting on i1 and R primer sitting on v (generating a product of about 5000, provided that F primer is very close to the beginning of i1, e.g. using the same primer you used for cloning).
Good luck!
Hi Homa, Hope you have got your clone. If not, here are some steps I would take:
1. Use NEB's high fidelity restriction enzymes for cloning if possible. BamH1 could be a tricky one. But HF BamH1 is very helpful.
2. Purify your PCR products with columns, measure the concentration. Digest
Hi Xiaochong, whatever you mentioned is exactly what I have done so many times. I am using fast cloning kit for ligation (T4 ligase) which can be done in 5 min however I extend it to 1 hour. Still it didn't work. I have used T4 for cloning for the same genes before and it always worked. The only difference is that last time my genes were already in other vectors but this time I am amplifying with PCR.
Hi Homa,
-I used to use Pfu polymerase for PCR since its accurate than Taq polymerase.
- Instead of using ligation kits,try using T4 ligase for 16 degrees temperature over night.
- Try using different strain of E.coli (designed for high expression of protein). If it is a human protein then you should go for strains designed specifically for expression of human proteins. I had the same problem When I was trying to express fusion protein.
It is always advisable to clone in good transforming strain and get protein expressed in modified strains.
Dear Homa, Were you able to amplify your genes of interest from the colonies you observed? and what was the product size?
Dear Priyanka, thanks for the comment but can you give me more details. I am using T4 ligase, but I guess you are suggesting me to extend ligation time for overnight. But what degrees you are suggesting?!!
I am using top 10 (invitrogen) very efficient expressing host.
Vinoth, yes I got bands for some samples. I am using detection primer and gave me the size of 800 bp.
From your answer, I can conclude that the detection primer included gene specific regions?
Dear Homa,
Please let me know which bacterial strains you are using for transformation of final vector and what method you are using for transformation.
Yes vinoth.
Manu, i am using top10 from invitrogen, highly efficient compete cells and heat shock method.
I'm wondering that this problem can be similar to mine. During my M.Sc. course, one of my mini-prep rates was so low similar to your's. I repeated mini-prep multiple times but the results was similar. I tested every thing but all seemed to be ok! I was not the only person that faced this problem. That problem never solved but after repeating the transformation of ligation products with a new competent source, every thing was fine. This may solves your problem too. If your vector in low copy number, you may use chloramphenicol along with kanamycin when culturing transformed strain. This may increase mini-prep rate but repeating the transformation is the best choice.
Dear Homa, Try using vector control (undigested and digested) to check for self ligation of your vector...
Dear Homa, try the DH10B strain - it is optimised for big constructs. Alos for big fragments use ratio of 1:1 (molar). If it is not working again, clone the intermediate fragment (the 2 genes) in cloning vector like pUC19, confirm the sequence of the 5' and 3' ends and then subclone.
Dear Homa,
I agree with Slavil. You should try DH10B starin and also modify your transformation protocol or you can try electroporation method also.
If it is not working again, clone your gene into any cloning vector first confirm the sequence and then subclone it.
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