Knight:Colony PCR protocol

Solutions/reagents: PCR supermix VF2  (40 µM)  VR  (40 µM) colony template Equipment: ThermocyclerElectrophoretic unit</li>Reaction tubes</li></ul> Steps: <ol>  Reaction Mixture  Use the following table as a checklist for preparing the reaction in reaction tube (1): </li>   PCR conditions  Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation Denature: <font color=#357EC7>95°C , <font color=#357EC7>15 mins </li></ul>Thermocycling No. of cycles: <font color=#357EC7>39 </li>Denature: <font color=#357EC7>94°C , <font color=#357EC7>30 secs </li>  Anneal: <font color=#357EC7>56°C , <font color=#357EC7>30 secs </li> Elongate: <font color=#357EC7>68°C , <font color=#357EC7>60 secs </li></ul><font color = "#800517">''Elongation time : 1 min per kb of expected product. I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.'' TerminationElongate: <font color=#357EC7>60°C , <font color=#357EC7>20 mins </li>Hold: <font color=#357EC7>4°C , until removed from machine </li></ul></li> Perform agarose gel electrophoresis of appropriate quantity of  PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 55 mins