Simply Cloning - Chapter 5 - Insert Restriction Digest

Описание: During insert restriction digest we are going to prepare the PCR fragment for ligation by creating sticky ends with the same pair of restriction enzymes that we used for cutting the vector.

The protocol for insert restriction digest is very similar to that of vector restriction digest:

1. Prepare restriction mix
o 44 µl gel purified insert
o 5 µl 10X enzyme buffer
o 0.5 µl restriction enzyme A (5 units)
o 0.5 µl restriction enzyme B (5 units)
2. Mix up by pipetting up and down
3. Incubate at 37 oC for 45 min

For the insert restriction digest we will need the gel purified PCR fragment, NEB Buffer 3, an empty Eppendorf tube, and our restriction enzymes, which are XhoI and BamHI.

In the Eppendorf tube I am going to combine:
• 44 µl gel purified PCR product
• 5 µl of NEB Buffer 3
• 0.5 µl of XhoI
• and 0.5 µl of BamHI
And I will mix it up by pipetting.

I will incubate the insert rectriction digest in a 37 oC incubator for 45 min.

At the end of insert restriction digest we have to deactivate the enzymes so they don't interfere with ligation.

In most cases it could be done by heat deactivation. I prefer to use my gel purification kit, which in this case serves as an enzyme removal kit.

To clean the digested insert on with the gel purification kit I add the capture buffer directly to the tube with insert digest and proceed with the protocol you saw in the previous step.