184.108.40.206. Cloning full-length viral RNAs
Cloning of long (~10 kbp) PCR products into plasmid vectors may be not very efficient. We recommend to use topoisomerase pCR-XL-TOPO cloning which is vector specifically designed for cloning of large products. This vector require 3’ terminal A overhangs in the PCR products, therefore the first stage is addition of the 3’ overhangs to the Phusion- generated blunt ends.
1. Mix the following:
1.1. 50 μl purified PCR fragments,
1.2. 6 μl 10x Taq polymerase buffer,
1.3. 3 μl 10mM dNTPs,
1.4. 1 μl Taq polymerase.
2. Incubate at 72°C for 10 min.
3. Separate the fragments by electrophoresis in 0.8% agarose gel in TAE buffer.
4. Stain the gel with crystal violet (see InVitrogen TOPO XL PCR cloning kit).
5. Excise the 10 kb full-length cDNA fragments.
6. Extract DNA using the Gel Purification reagents included in the TOPO XL PCR Cloning kit (Invitrogen).
7. Ligate fragments into the pCR XL TOPO vector by mixing:
7.1. 4 μl of the purified product (approximately 10 to 50 ng)
7.2. 1 μl of pCR XL TOPO vector.
8. Incubate reaction for 5 min at 25°C.
9. Stop reaction by addition of 6x TOPO cloning Stop Solution.
10. Mix for a few seconds at room temperature and place the reaction to ice.
11. Proceed immediately to transformation of the supplied One Shot competent cells (Invitrogen), following the manufacturer’s instructions.