Step 1. PCR amplification of insert with a high fidelity polymerase
Using a high fidelity polymerase, Q5 or Phusion, amplify the desired DNA insert.
Step 2. Gel isolation and Quanitification of insert
The gel isolated product must be high enough concentration to proceed to the next step.
Step 3. Using Taq polymerase to add an adenine nucleotide to the 3′ side of insert
In a 25ul reaction add 0.15 pmoles* of the DNA insert with the following reagents:
2.5ul 10X NEB Standard Taq Polymerase buffer
0.5ul dATP (10mM)
0.2ul Taq Polymerase (NEB 5U/ul)
X ul PCR gel isolated DNA insert (0.15 pmoles)
(25-3.2-X) ul H20
– Add mineral oil to the reaction
– Incubate reaction at 72C for 20 minutes
* This amount depends on the size of the insert. For example, 0.15pmoles of a 1,000bp product is 97.5ng. You can use this molarity calculator to help you determine the correct amount.
Step 4. PCR2.1 Topo Reaction
See the Topo TA cloning manual for information
In summary, add enough insert for a 3:1 ratio of insert:vector. You must calculate the concentration of the insert from step 3. The vector PCR2.1 topo is 3,900bp and supplied at 10ng/ul. You will use 1ul of PCR2.1 topo which is 0.000392 pmoles. With your calculated insert from step 3, assemble the topo reaction. The topo ran is 6ul = 1ul PCR2.1 topo + 1ul salt solution + Xul insert from step 4. + Y water. Incubate the reaction at 22C for 5-30 minutes. 30 minute incubations are for maximal efficiency.
Step 5. Transformation of topo reaction
Follow the standard competent cell protocol and use 3ul of the completed topo reaction to 30ul of competent cells. From the 300ul outgrowth, plate 100ul of this onto the LB agar plates containing ampicillin for bacterial selection and grow at 37C for 16 hours.
Step 6. Screening colonies
Pick 10 colonies from the bacterial plate and grow up for plasmid DNA miniprep
Step 7. plasmid DNA miniprep and restriction digest
Perform DNA miniprep purification protocol and digest the DNA to ascertain if there is an insert of the expected size. There are (2) EcoR1 sites that flank either side of the insertion site and may be used id appropiate.
Step 8. DNA sequencing
Send each clone that displays an insert of the correct size for DNA sequencing