Entry Vector Cloning Protocol
The open reading frame (ORF) of the clone must be PCR amplified in order to clone the ORF into an Entry Vector. Please view the Primer Design and PCR Amplification of ORFs Protocol before moving forward.
Step 1, Electrophoresis:
- Confirm the size of the amplification product by agarose gel electrophoresis and purify the remainder of the reaction using a purification column or similar method.
Step 2, Digestion:
- Elute the DNA from the purification column in 26 μl of 10 mM Tris buffer. Set up a digestion reaction as described below, substituting other restriction enzymes as appropriate.
| Component | Volume |
|---|---|
| 10X restriction buffer | 3 μl |
| Sgf I (10U/μl) | 0.6 μl |
| Mlu I (10U/μl) | 0.6 μl |
| Purified PCR product | 26 μl |
| Total volume | ~30 μl |
Mix well, and incubate at 37°C for 3 hrs.
- Purify the digestion reaction using a purification column and elute in 18 µl of 10 mM Tris buffer.
- Quantitate the DNA by UV at A260.
- Digest the pCMV6-Entry vector with the restriction enzymes corresponding to the sequences added to the ORF. The pCMV6-Entry Vector is available from OriGene as 10 µg lyophilized DNA (Cat# ). Resuspend the lyophilized DNA in 100 µl dH2O, and incubate for at least 30 min before use. Set up a digestion reaction as described below, substituting other restriction enzymes as appropriate.
| Component | Volume |
|---|---|
| 10X restriction buffer | 3 μl |
| Sgf I (10U/μl) | 0.8 μl |
| Mlu I (10U/μl) | 0.8 μl |
| Nuclease-free water | 15.4 μl |
| Vector DNA | 10 μl |
| Total volume | 30 μl |
Incubate at 37°C for 3 hrs, then add 1 μl antarctic phosphatase (units used according to the manufacturer’s protocol), and continue the incubation at 37°C for another 30 min. Dephosphorylation of the digested vector is essential to eliminate self-ligation.
- Purify the desired vector fragment by running the digestion reaction on an agarose gel, and isolating the appropriate band using a gel purification column. Elute the digested plasmid vector in 40 μl of 10 mM Tris buffer.
Step 3, Ligation:
- Set up a ligation reaction with the purified vector and insert fragments:
| Component | Volume |
|---|---|
| 10X ligase buffer | 1μl |
| Nuclease-free water | 3.5μl |
| T4 DNA Ligase | 0.5μl |
| Vector Fragment | 2μl (approx. 10ng)* |
| PCR product | 3μl (approx. 30ng)* |
| Total volume | 10 μl |
Incubate the ligation reaction at room temperature for 30-60 minutes. * Alternate ratios may need to be tested to obtain optional ligation efficiency.
Step 4, Transformation:
- Transform 1 µl of the ligation mixture using 20 µl high efficiency competent E. coli cells (ideally 1x108 CFU/ug). Following transformation, resuspend cells in 200 uL LB.
- Plate the entire transformation reaction on a standard LB-agar plate containing 25 µg/ml kanamycin. Incubate at 37°C overnight.
- Pick at least 4-8 independent colonies to do miniprep from each ligation. Confirm the insert by restriction digestion and/or vector primer sequencing.