Topo Cloning
(per Adam 8/2004)
Topo Cloning is a procedure that is used to amplify a small amount of a single DNA fragment. This is done by cloning the fragment into a vector, transforming E. Coli cells, and using blue/white selection to determine transformants. Once a transformant has been isolated, a culture can be inoculated and the Qiagen Plasmid Prep protocol can be used to isolate the plasmid containing the cloned copies.
1. Isolate PCR product from the gel following Qiagen Qiaquick Spin protocol.
(a) Excise DNA fragment from gel.
(b) Add 3 volumes of buffer QG to 1 volume of gel.
(c) Incubate at 50°C for 10 minutes, vortex to mix.
NOTE: The color of the solution must be yellow
(d) Add 1 gel volume of isopropanol and mix.
(e) Apply sample to QIAquick spin column, centrifuge for 1 minute.
(f) Discard flow through.
(g) Add 750µL buffer PE, centrifuge for 1 minute.
(h) Discard flow through and repeat wash.
(i) Centrifuge column for an additional 1 min to remove residual buffer.
(j) Transfer the column to new tube and add 30µL buffer EB
NOTE: The EB should be preheated to 65°C.
(k) Let column stand for 1 min and centrifuge for 1 more min.
2. Combine 2µL DNA, 1µL salt solution (supplied in the Invitrogen Topo Cloning Kit), and 2µL sterile H2O (also found in kit). Spin contents briefly so that they are mixed in the bottom of the eppendorf tube. (For lower concentrations of DNA, you can use 4µL DNA with no extra H2O.
3. Add 1µL Topo vector (found in kit) directly to mixture.
NOTE: It is crucial to keep the Topo vector on ice at all times. The Topo vector self-catalyzes the ligation reaction via topoisomerases, when left at room temperature these enzymes lose activity.
4. Allow ligation reaction to proceed at room temperature for 5 minutes.
5. Add chemically competent DN5α E. Coli cells (or TOP10 competent cells supplied with the kit) to Topo mixture.
NOTE: A tube of cells (~100µL) can be split 3 or 4 ways.
6. Place topo/cells mixture on ice for ~20 minutes.
7. Heat shock the mixture at 42°C for 30 seconds then return to ice.
8. To allow cells to recover, add 250µL LB (or SOC) and spin for 1 hour at 37°C.
9. Prepare plates for plating cells
(a) Place LB Kan50 (or LB Carb50) plates upside down in 37°C incubator for 20 minutes to dry them.
(b) Add 40µL IPTG (20%) and 80µL X-Gal (20mg/mL) to plates and spread with hockey stick.
NOTE: If TOP10 cells are used, IPTG is not necessary.
(c) Let plates stand at room temperature until cells have recovered.
10. Plate 200µL of the recovered cells per plate. However, if transformation efficiency is low plate entire contents of tube. (IF too high, plate less.) Colonies that have acquired the cloned product/topo vector construct will appear white on the plates. Blue colonies are ones that have NOT acquired the product/vector.
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