Added dATPs to the ends of untreated and g034 treated Luc14 P197/198 PCR products
- From the manual:
- After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase. Incubate the reaction for 10–15 minutes at 72°C and use in the TOPO® Cloning reaction.
| 0.5ug PCR product||6ul
| 5x longamp taq buffer||2ul
| 10mM dATP||1ul
| longamp taq||0.5ul
Incubated at 72°C for 15 min in PCR machine
Cut with PfoI for 3:12 hours at 37°C in heat block
| 0.5ug PCR product+dATPs||10ul
| 10x optizhyme buffer 4||2ul
Heat inactivated in PCR machine, 65°C for 20 minutes.
- Set up the TOPO Cloning reaction
- Mix the reaction below gently and incubate for 5 minutes at room temperature (22°C to 23°C)
- Decided to do two reactions for each samples, one wiht .5ul and one with 4ul of PCR product
| Fresh PCR product ||0.5 or 4 µL
| Salt Solution ||1 µL
| Water ||add to a total volume of 5 µL
| TOPO® vector ||1 µL
| Final Volume ||6 µL
Incubated for 30 minutes at room temp.
- Notes from the manual:
- Note: The red or yellow color of the TOPO® vector solution is normal and is used to visualize the solution.
- A Salt Solution (1.2 M NaCl; 0.06 M MgCl2) is provided to adjust the TOPO® Cloning reaction to the recommended concentration of NaCl and MgCl2... We have found that including salt (200 mM NaCl, 10 mM MgCl2) in the TOPO® Cloning reaction can increase the number of transformants 2- to 3-fold. In addition, incubating the reaction mixture for greater than 5 minutes in the presence of salt can also increase the number of transformants... Including salt in the TOPO® Cloning reaction allows for longer incubation times because it prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating from the DNA. The result is more intact molecules, leading to higher transformation efficiencies.
- For most applications, 5 minutes will yield sufficient colonies for analysis. Depending on your needs, the length of the TOPO®-cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (greater than 1 kb) or if you are TOPO®-cloning a pool of PCR products, increasing the reaction time will yield more colonies.
- You may store the TOPO® Cloning reaction at −20°C overnight.
- Place the reaction on ice and proceed to Transform One Shot® competent cells
- warmed LB plates for 30 40 minutes
- added 2ul and 4ul of each of the four reactions to 50ul of cells
- incubate on ice for 10 minutes
- grow overnight at 37 in wang lab incubator
Note: It is essential that LB plates containing ampicillin are pre-warmed prior to
1. Add 4 µL of the TOPO® Cloning reaction from Set up the TOPO® Cloning
reaction, step 2 on page 13, into a vial of One Shot® chemically competent
E coli and mix gently. Do not mix by pipetting up and down.
2. Incubate on ice for 5 minutes.
3. Spread 50 µL of cells on a prewarmed LB plate containing 50–100 µg/mL
ampicillin and incubate overnight at 37°C.
4. An efficient TOPO® Cloning reaction should produce several hundred
colonies. Pick ~10 colonies for analysis (see Analyze positive clones on