Haynes Lab:Notebook/CRISPR Editing/2015/01/29

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Topo cloning

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.
thing amount
0.5ug PCR product 6ul
5x longamp taq buffer 2ul
10mM dATP 1ul
H20 .5ul
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

thing amount
0.5ug PCR product+dATPs 10ul
H20 18ul
10x optizhyme buffer 4 2ul
PfoI 1ul

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
Reagent* Volume
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
  • Transform
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 spreading. 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 page 20).