 |
Last updated |
| August 10, 2003 | |
         |
 |
 |
|
 |
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 |
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