Over the last few months I have had serious problems in cloning as a result of too much colony in control plate. Here I am explaining what I did:
I was trying to clone a 1.2 kb and 3.4 kb insert into a vector which is about 9.1 kb in size. I isolated my insert from its parent vector through digestion with AsiSI (from NEB) and also the recipient vector was digested with the same enzyme. I cut 5 µl of miniprep DNA in a 20 µl reaction using 2 µl of AsiSI. I incubated the reaction mixture at 370C for 4 hrs. After incubation, I dephosphorylate the vector with 1 µl of TSAP (from Promega) and heat inactivated the TSAP at 740C for 15 mins. After that, I set up ligation along with two controls, one with ligase and another without ligase. I got approximately the same amount of colony in all the plates. Then I checked at least 100 colonies from the plate that was transformed with ligation mixture but got no positives. I repeated the cloning several times and every time, I got the same result. Then I planned to determine the amount of background colony. For doing this, I again cut the vector and dephosphorylated it and set up two reactions, one with ligase and the other without ligase in 10 µl of reaction. Then I transformed this 50 ng vector with ligase and without ligase along with 50 ng uncut vector. What I saw the next morning was exactly the same amount of colony in all the plate. I don't know what the problem is. The enzyme is clearly cutting the insert from its parent vector quite nicely. I repeated the cloning with every option that I know, like incubating the digestion reaction for 1 hrs, 2 hrs, 3 hrs, 4hrs, setting up the ligation at 40C, 160C, gel purifying the vector, PCR cleaning the vector. Can you please suggest what the strange events happening here could be the result of? I would be grateful to all of you.
Thanks a lot Ermelinda for your suggestion. As noted by NEB AsiSI has star activity and thats why I did not incubate the digestion reaction overnight. I incubated maximum for 4 hrs.
Md. Hassan,
What plates are you using for this experiment of yours. As in with what Antibiotic? Also, are you sure that the antibiotic resistance gene in your insert/ vector is being retained intact after the digest.
Since your plate without ligase is also showing the same number of colonies, there is something seriously wrong with your selection of colonies.
I would also think about uncut vector: Run the vector on a gel, cut out the band of interest (digested vector) and purify it. This usually solves the problem of uncut vector.
The other possibility. Which antibiotics are you using in which concentration? And how old is your stock solution?
Hi,
you may consider a preselection digest:
Description and design at preselector dot org
Best
Ralf Mrowka
Hi, I suggest to test also the competent cells. Try to plate the untransformed cells onto your dishes with the antibiotic to make sure that those cells are not resistant
Did you excise the bands of interest for both vector and insert, and then purify?
I recommend to check the phosphatase, first. Cut the backbone, treat it with ligase and see by gel or transforming bacteria. Some times this is the problem, and your backbone is being religated spontaneously.
Another reccomendation would be run the backbone and the insert just before the ligation reaction. Here you should see single bands.
Finally, check the antobiotic in your plates is not degraded! (ampicillin should be poured at
I would agree with @Christian Praetorius. You should go gel extraction of band of your interest. Reason is very simple that even if you dephosphorylate, there is ample chance of uncut plasmid which is being transformed. Digest your sample, do gel extraction and purify the DNA. Dephosphorylate the sample, heat inactivate and transform. You will get positive colonies. Better option would be use two enzymes, that would ensure unidirectional cloning. Best of luck
You clearly must have a significant amount of uncut vector in your prep, or alternatively if you cut your fragment out of a small vector you may also have some contamination from the fragment isolation. If the second is true you will get colonies when you ligate the fragment by itself. This would not explain your high background on the vector alone, but may be something to consider.
Also double check that when you purified your fragment you did so using the correct UV setting on the transiluminator since the DNA can be seriously damaged using the analytical setting as opposed to the preparative setting.
It seems like you have already gotten some pretty good feedback on how to get rid of uncut supercoiled vector from your vector prep, but in my opinion that is likely your problem. Gel purfication of your cut vector should resolve that problem.
AsiSI is not DAM or DCM methylation sensitive and since it came out of a vector I assume it had previously been grown in E. coli, but that is something else to look for when you have poor cutting.
Good luck
Good luck
Thank you all for some nice suggestion. To Ameya and Christian, I am using Amp plate (Concentration 100 microgram/ml) and I made a fresh stock.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology