Use Qiagen PCR Cleanup Kit to remove dNTPs and polymerase that would fill in sticky ends.
Example double digest
plasmid PCR product DNA 10 ul (2.7 ug) 20 ul (unknown #ug for now) Buffer 2.5 ul 10X NEB2 2.5 ul 10X NEB2 Enzyme 0.5 ul EcoRI 0.5 ul EcoRI Enzyme 0.5 ul XbaI 0.5 ul XbaI __________________________________________________________ H2O to final volume of 25ul (not counting volume of enzyme)
^New England Biolabs sells buffers to use with their enzymes. You will use their buffer called NEB2.
- Assemble the reactions in eppendorf tubes in the following order
- DNA (you can get pCX-NNX from the teaching faculty)
- Enzymes (you can get these from the teaching faculty)
- It is very important to be thoughtful to your labmates here!
- Use only clean pipet tips when going into the lab stocks of plasmids and enzymes.
- Keep the enzyme stocks cold or they will denature and be useless for everyone.
- Flick your tubes to mix the contents, give them a quick spin in the microfuge if there are droplets stuck in along the wall and incubate the digests at 37°C for the remainder of the lab period.
Plasmid digests will be finished almost instantly but PCR products are harder for enzymes to digest and should be allowed to react for at least one hour, in some cases overnight.
- Qiagen reagent with Guanidine HCl
- Qiagen reagent with Ethanol
- Qiagen reagent = 10 mM Tris, pH 8.5
- NEB Buffer2 (10X)
- 100 mM Tris-HCl, pH 8
- 100 mM MgCl2
- 0.5 M NaCl
- 10 mM DTT
- BSA can be added if enzyme limiting