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You open the incubator in the morning and to your dismay there are a hundred glorious colonies… on your vector-only control plate.
This has happened to me more times than I wish to admit. While there are a number of potential causes, I’ll highlight a few of the more likely culprits and their solutions.
Did you completely digest the vector?
If the vector is not completely digested, it is very easy to end up with intact vector—sans your insert—in your transformants.
To solve this issue, ensure that you are using the correct buffer for your restriction enzymes, allow sufficient time for the digestion to occur, and make sure you have active enzymes.
Use the correct buffers with the correct additives!
Many brands of restriction enzymes offer easy-to-use calculators to determine the best conditions for the enzymes you are using. Thermo and NEB both offer such calculators. Additionally, some enzymes work better when BSA is added. Don’t forget to find the most compatible buffer.
In the case of double digests, you may stumble upon a scenario in which no buffer is compatible with both enzymes. In this case, you’ll need to do the digests sequentially. As long as you are going to purify the DNA between digests, it doesn’t matter which enzyme you start with.
The introduction of universal buffers has made double digests easier. Universal buffers, such as the CutSmart buffer from NEB, are convenient and work with many enzymes. However, there are still enzymes that work better with a more specific buffer and are either incompatible or less active with the universal buffer. Make sure you check the recommendations of the manufacturer of your enzymes.
Most enzymes will cut 1 ?g of DNA in 1 hour at 37°C. However, you should check the specifications of the enzyme(s) you intend to use. The High Fidelity enzymes from NEB can digest 1 ?g of DNA in as little as 5-15 minutes.
Intuitively, it may seem that the longer you leave your digest reaction at 37°C, the more complete your digest will be. However, digesting your DNA for too long can result in non-specific cuts or other alterations of your DNA template. This unwanted activity is called Star Activity and can damage your DNA template.
I don’t know about your lab, but I have found tubes of enzymes that expired a decade ago. In a pinch, I’ve used these enzymes, and they have worked. But don’t waste your time unless you are unable to acquire new enzyme.
How do you know if your enzyme is working? If you are doing a double digest with two unique enzymes, set up single digests simultaneously as well as an undigested sample. When you run these on a gel, you should be able to see that the linearized band is running at a different size than the undigested vector. If this is true for only one of your single digests, then the other one is probably the culprit and should be replaced.
How did you purify your digested vector?
If you are running your digested vector on a gel, be sure to run the gel long enough to get sufficient separation of your bands. If you fail to eliminate undigested vector, you will get bacterial colonies that have a fully intact vector, without containing your insert. By fully separating the digested vector from the undigested, you can eliminate the issue of an incomplete restriction digest. However, it may be beneficial to have controls on this gel to ensure you are excising the correct band. Run an undigested sample as well, so that you can clearly tell which band is digested and which is not.
Helpful Hint: Do not overload your wells! If you digest 20 ?g of DNA and load it all into a single lane of an agarose gel, you are likely to get a smeary mess. This makes it nearly impossible to excise the digested vector for purification! Run multiple digests, or at least separate the sample into multiple lanes to alleviate this problem.
Did you dephosphorylate the vector?
Phosphatases remove phosphates from the 5’ and/or 3’ end of a cut DNA molecule, inhibiting re-ligation of your digested vector. Dephosphorylation of your vector can be crucial, especially if doing blunt-end cloning or using a single restriction enzyme—all times in which the vector can self ligate, without your precious insert.
There are a variety of phosphatases available:
- Calf Intestinal Alkaline Phosphatase (CIP)
- Shrimp Alkaline Phosphatase (rSAP)
- Antarctic Phosphatase
- Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase
Each of these phosphatases has pros and cons. For instance, Antarctic Phosphatase and Shrimp Alkaline Phosphatase can be inactivated by heating at 65°C, while Thermo’s FastAP can be inactivated at 75°C. This ensures that the phosphatase is not enzymatically active and carried over into the ligation step.
Helpful Hint: If using a phosphatase that can be heat inactivated, take the time to do this step to ensure there is no carry-over of active phosphatase to the ligation step. If using CIP, or another phosphatase that cannot be heat inactivated, be sure to only use as much enzyme as necessary to minimize carry-over into the ligation step. Additionally, gel isolation after dephosphorylating the vector should prevent carry over.
Did you use the right plates?
This may seem trivial, but in a lab you sometimes have to rely on your colleagues to correctly prepare and label solutions, plates, lab supplies, etc. Ensure that you are using the proper plates with the appropriate growth medium and antibiotic concentration!
I hope these solutions help you get the cloning ratios you desire. Happy Cloning!