Restriction Enzyme Digest of Plasmid & Insert DNA
Equipment and Consumables:
Heat Block or Incubator at 37°C
Vortex
Restriction Enzyme #1 (and #2)
These are often extremely heat sensitive. They will be in glycerol stock and thus do not need hand thawing. Move immediately from the freezer to the ice box.
Appropriate Restriction Enzyme Buffer
For double digests, it’s OK to use a buffer which gives 100% activity for one enzyme, and 75% activity of the other, but lower than this is not good. Also check the appropriate digest temperature, it’s usually 37°C, but not always - always double check the ideal digest conditions for your chosen enzyme.
Can be hand thawed
~250 ng Plasmid DNA
Try to quantify via gel comparison to a known standard or alternatively use a nanodrop, ensuring you blank with the correct storage buffer.
Range may be 100-1000 ng of each DNA, in a volume of 30 – 200 µl.
~250 ng Insert DNA
This is relatively easy to calculate if you’re digesting a quantified sample that you paid for. If this is a PCR sample that you produced yourself, try to quantify via gel comparison to a known standard or with a nanodrop.
Range may be 100-1000 ng of each DNA, in a volume of 30 – 200 µl.
Ice in ice box
For Purification:
Centrifuge (Ideally one capable of 10,000g)
Wash Buffer 1 (PB): 5 M guanidine HCl, 30% isopropanol. Not sterilised.
Wash Buffer 2 (PE): 10 mM Tris-HCl, pH 8, 80% ethanol. Not sterilised.
Elution Buffer (EB): 10 mM Tris-HCl, pH 8. Autoclaved.
Note: The plasmid and insert digests are described separately and sequentially below for clarity, but these can and should be set up more-or-less at the same time;
Plasmid Digest Protocol:
Calculate how much volume of plasmid you need to use to get 250 ng – call this ‘X’, and calculate how much water to add to the digest, call this ‘Y’; that is Y = (100 – 10 – 2 – X).
Retrieve 10x restriction buffer from freezer, thaw completely, and vortex to mix. The same tube of buffer can be used many times, if you are careful with your aseptic technique.
Retrieve the plasmid from the freezer, allow to thaw, (e.g. in 37°C water bath, or rub in your hands, or on bench etc), then put it on ice when it is thawed. Its not good to leave the plasmid stock at room temp or above for prolonged periods or it may degrade due to traces of nucleases.
Retrieve the restriction enzyme(s) from the freezer, put IMMEDIATELY on ice. These are heat-sensitive and you need to look after them. Do not leave them at room temp. Keep on ice while setting up the reaction, then immediately put back in freezer. These don need to be thawed, they are in a glycerol solution which doesn’t freeze at -20°C.
Label your tube(s), then set up the digest(s) by adding the ingredients in the following order. Make sure you use excellent aseptic technique, and change tips every time. Its OK to set this up at room temp, the reaction tube doesn’t have to be on ice.
- 10 ul of 10x restriction buffer
- ‘Y’ µl of sterile MQ water
- ‘X’ ul of plasmid DNA
- 2 µl of (each) restriction enzyme
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Total 100 µl
Mix by flicking, then tap on bench to get liquid to bottom of tube. Incubate at correct temperature for approx. 2 hours. (1 - 4 hr is OK, but overnight digest is too long; this can lead to ‘raggedy ends’ of the plasmid even if it looks OK on a gel; this is due to non-specific nuclease activity)
100 µl seems like a large volume to use, but this helps dilute any impurities in the plasmid prep. Increasing the total volume of the digest and/or reducing the volume of plasmid added often help to improve the quality of a poor digest.
If the digested plasmid is for the purpose of ligation, we can use a large volume, since it will be column-purified & concentrated anyway.
Insert Digest Protocol:
Calculate how much volume of insert DNA you need to use to get 250 ng – call this A, and calculate how much water to add to the digest, call this B; that is B = (100-10-2-A).
Label your tube(s). Set up digest by adding ingredients in the following order. Make sure you use excellent aseptic technique, and change tips every time. Its OK to set this up at room temp.
- 10 µl of 10x restriction buffer
- ‘B’ µl of sterile MQ water
- ‘A’ µl of insert DNA
- 2 µl of (each) restriction enzyme
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Total 100 µl
Mix by flicking, then tap on bench to get liquid to bottom of tube. Incubate at correct temperature for approx. 2 hours. (1 - 4 hr is OK, but overnight digest is too long)
OPTIONAL: Dephosphorylate the vector by adding 11 µl of 10x phosphatase buffer and 2 µl of antarctic phosphatase enzyme, incubate 30 min at 37°C.
This step prevents the vector religating to itself; it is not required (in theory!) when you are cloning with two different restriction enzymes. Only buy the reagents to perform this step if you Really need to do a single restriction enzyme digest.
You can now proceed to the Ligation Protocol, but the chance of success is quite low. It can be significantly improved by adding either an inactivation or a purification step here.
Heat Inactivation Protocol: You can use heat to inactivate the restriction enzymes and hope that they don’t get in the way of the ligase. Some restriction enzymes don’t inactivate at all however, so you can also perform the…
Column Purification Protocol: Similar to the tail end of the miniprep protocol, you can use PB, PE and EB buffers to purify your digested DNA fragments from the enzymes used to cut them.
Agarose Electrophoresis Purification Protocol: Safer alternative without Guanidine buffers. This is identical to the protocol performed to purify a PCR fragment. Follow the purification steps at the bottom of the Agarose Electrophoresis protocol.
Acknowledgements:
Coleman Protocols 2017 + 2019 http://coleman-lab.org/