I need to insert a 9kb fragment into a 11 kb vector. I planed to use gibson assembly and the enzyme I used was from clonesmarter(Seamless Assembly cloning kit) and Yeasen(Universal One Step Cloning Kit). I seperated my 9kb fragment into three 3k fragments and cut my vector with KpnI-HF. According to snapgene, the primers designed for these three fragments could work well and gave me my 20kb vector. I followed the manual from the company(50 ℃,15 min) and transformed the reaction liquid into Mach1. I got seven transformants and got plasmids. However, the size of all 7 plasmids were wrong(show in the picture and the right lane was my 11 kb vector without cut, and the brightest band of marker was 5k). I don't know what happened, could anyone help me? Thank you very much!
Hello!
I have been doing very similar cloning for the past months. I have a 9kb insert separated into three fragments with 20bp overlapping regions to perform Gibson assembly.
If you are happy with how you designed the overlapping regions and primers and it works on Snapgene then I can give you the advice that worked for me when it comes to the actual cloning.
First, I'm guessing you are amplifying your three fragments by PCR, do you run them on a gel and do a gel extraction or do you just do a PCR clean-up straight away? If you are just doing a PCR clean-up with no gel extraction then a lot of non-specific amplicons could also be purified along with the actual 3kb amplicon. These non-specific amplicons contain the homology region that Gibson will use (because the primers used to amplify them have the homology region as well), and because they are shorter they will be incorporated into your vector more efficiently than your actual 3kb fragment. If you haven't done it yet, run a gel with your PCR product without PCR purifying it and see if there are any non-specific bands, if there are non-specific bands then run all your PCR samples on a gel (without previously purifying them) and gel extract the appropriate band. If your PCR is super neat and there are no non-specific bands then this might not be necessary but I would still recommend it.
The problem with doing a gel extraction instead of a PCR purification is the high amount of impurities that the extracted DNA has (salts, etc.) these will inhibit the Gibson reaction. To fix this you can do a PCR purification on the gel-extracted DNA. Although you will lose DNA quantity, not necessarily DNA concentration if you take care to dilute it in less water. This step is critical.
You can also digest the backbone for longer periods or overnight and also run it on a gel for longer to make sure you reduce the number of undigested contaminants as much as you can when extracting it. Remember to also PCR purify your gel-extracted backbone!!
When doing the Gibson assembly, you can run it for 4 hours instead of 15 minutes which can increase efficiency. I used Gibsons assembly from NEB, then changed to their HiFi assembly mix which is essentially the same but better, and it worked for me.
Hope you manage to clone it!
Bruno Salomone Gonzalez de Castejon Thank you for your advice. I got my fragments from gel extraction.
I agree with the cloning recommendations. After transformation and plasmid isolation, I would also linearize the plasmids by restriction enzyme digestion before running them on a gel to avoid those supercoiled bands you are getting. Also, it is quite difficult to assess different sizes in the 10-20kb range in an agarose gel unless you are performing PFGE, so another recommendation is to digest your plasmid with a restriction enzyme that would render a unique restriction pattern of 0.5-8 kb fragments (easier to discriminate in a regular agarose gel), so that you know your fragments are there and also to easily assess total vector size by summing up the size of the individual restriction fragments.
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