#Noti enzyme how to#

Because of this we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples.

When running a gel for purification purposes it is important to have nice crisp bands and to have space to cut out the bands. Run your digested DNA on an agarose gel and conduct a gel purification to isolate the DNA.

Isolate your insert and vector by gel purification:.

CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are commonly used. You should treat your digested recipient plasmid with a phosphatase prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose. If you are going to use only one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent re-circularization of the recipient plasmid. It is also critical that as much of the recipient plasmid as possible be cut with both enzymes, and therefore it is important that the digest go at least 4 hours and as long as overnight. We recommend 1.5-2μg of donor plasmid and 1μg of recipient plasmid. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting material. Set up restriction digests for your donor and recipient plasmids. If you select enzymes that can function in the same buffer, it will save you time in future steps. New England Biolabs for more information about restriction enzyme buffers). If you are lucky enough to have multiple options for enzymes that flank your insert and will result in correct orientation in the recipient plasmid, it is useful to see if one set of enzymes will work in the same restriction enzyme buffer (see

Adding desired restriction sites to your recipient plasmid : You can modify the MCS of your recipient plasmid using Annealed-oligo Cloning.

However, you still need to avoid restriction enzymes that cut within your insert. This will allow you to produce a version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS.

Adding desired restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends of your oligos.

If you cannot find enzymes that meet these criteria, do not fear. It is also possible to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned in the correct orientation. Ideally, you will find two different restriction enzymes for your subcloning.

Are in frame with tags or fusion proteins in the recipient plasmid (if you are creating a fusion protein).

(You don't want to express the antisense version of your gene!)

Will result in your insert being in the correct orientation in the recipient plasmid.

Are in the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid.

Flank your insert, but do not cut within your insert.

When selecting restriction enzymes, you want to choose enzymes that: Using subcloning, you can easily move YGOI into a mammalian expression vector.Īddgene’s Sequence Analyzer, allow you to identify which restriction sites are present in a given sequence. The problem is that the only version of full-length cDNA you can find for YGOI is in a bacterial expression vector. You might need to express YGOI in cultured mammalian cells. Let's assume that you are beginning a new project on your gene of interest (YGOI for short). However, the same technique can be used to move promoters, selectable markers, or any other DNA element between plasmids.

#Noti enzyme how to#

For the purposes of this tutorial we will discuss how to move a cDNA from one plasmid to another. Subcloning by restriction digest is a commonly used lab technique. If you are not sure what vector to use, you can check out our The following technique can be used to easily move any piece of DNA from one vector to another as long as it is already bounded by restriction sites that are also present in the same orientation on your target vector.