PCR and Topo Cloning

[kiki06]

How easy is it to conduct PCR and then to use that product to subclone into TOPO TA vectors?

I have heard it is quite easy. Please post a short protocol also if you have one!

thank you very much
Kiki

[misteplix]

Originally Posted by kiki06

How easy is it to conduct PCR and then to use that product to subclone into TOPO TA vectors?

I have heard it is quite easy. Please post a short protocol also if you have one!

thank you very much
Kiki

TOPO Cloning [back]

Zero Blunt TOPO PCR Cloning Kit
(Invitrogen Life technologies)

This kit is specifically designed to clone blunt-end PCR products generated by thermostable proofreading polymerases such as Pfu.

1. PCR

Set up PCR reaction:

Add ddH2O to final volume to 49 ml
DNA template 1-5 ml
10 X reaction buffer 5.0 ml
dNTPs 5.0 ml
Primer 1 2.0 ml
Primer 2 2.0 ml
DMSO 3.0 ml
Add ddH2O to final volume to 49 ml
Hot Start
Add Pfu to the reaction 1.0 ml




Set up thermocyler program:
Hot Start
Denaturation 94°C 30 sec
Annealing 55°C 30 sec
Extension 72°C 90 sec (1 min/kb)
25-30 cycles

After cycling, incubate the reaction at 72°C for another 7 min
Cool the reactions to 4°C, ready for cloning
2. Cloning

At this point you should have your blunt-end PCR product ready for TOPO Cloning and transformation into the One-Shot TOPO10 Cells. It is very important to proceed as soon as possible from the TOPO Cloning reaction to transformation to ensure the highest cloning and transformation efficiencies

Cloning Reaction


Set up the following 5 ml TOPO Cloning reaction


Fresh PCR product 0.5 – 3.0 ml
Sterile ddH2O add to final volume of 4 ml
PCR-Blunt TOPO vector 1 ml
Final volume 5 ml

1. Mix gentaly and incubate for 5 minutes at room temperature (25°C). For the best possible results, don’t leave for more than 5 minutes or the transformation efficiencies will decrease.


2. Briefly centrifuge and place tube on ice. Reaction may be left on ice for up to 30 minutes. We recommend that you proceed immediately to the One-Shot Transformation Reaction.

3. Transformation

One-Shot Transformation Reaction

1. Add ~2 ml of the TOPO Cloning reaction into a vial of One-Shot cells and mix gently.
2. Incubate on ice for 30 minutes.
3. Heat Shock the cells for 30 seconds at 42°C without shaking.
4. Immediately transfer the tubes to ice and incubate for 2 minutes.
5. Add 250 ml of room temperature SOC or LB medium.
6. Cap the tube tightly and shake the tube horizontally at 37°C for 1 hour. Place on ice.
7. Spread transformation on a prewarmed LB plate with proper antibiotics and incubate overnight at 37°C.
8. Pick up colonies for analysis.

4. Analysis of Positive Clones

1. Take colonies and cultures them overnight in LB medium containing 50 mg/ml Kanamycin or 100mg/ml Ampicillin.
2. Isolate plasmid DNA using Promega mini prep method. (See protocol)
3. Analyze the plasmids by restriction analysis (EcoR I). Check by mini-gel.
4. For the plasmid which have right insert, continue cloning opa gene into expression vector.
5. Digest the plasmid and pET with NdeI/BamHI: Digest plasmid and pMAL c-II with BamHI only. (restriction enzymes used should be different for different constructions)
6. Dephosphorylation of expression vectors for 30 minutes.
7. Run a preparative gel to separate digested DNA fragments.
8. Cut the interested DNA bands and extract DNA from the gel by GeneClean Kit. (See GeneClean Protocol)
9. Set up ligation reaction.


Ligation Buffer 1 ml
Ligase 1 ml
Expression vector x ml
gene DNA y ml
Total volume 10 ml

Incubate ligation reaction at 4°C for overnight

[kiki06]

Awesome!

thank you so much misteplix!